Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity

Langdon, Casey G.; Gadek, Katherine E.; Garcia, Matthew R.; Evans, Myron K.; Reed, Kristin B.; Bush, Madeline; Hanna, Jason A.; Drummond, Catherine J.; Maguire, Matthew C.; Leavey, Patrick J.; Finkelstein, David; Jin, Hongjian; Schreiner, Patrick A.; Rehg, Jerold E.; Hatley, Mark E.

doi:10.1038/s41467-021-25829-4

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Published: 17 September 2021

Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity

Casey G. Langdon¹,
Katherine E. Gadek ORCID: orcid.org/0000-0001-5170-230X¹,
Matthew R. Garcia¹,
Myron K. Evans ORCID: orcid.org/0000-0002-7678-5815¹,
Kristin B. Reed^1,2,
Madeline Bush^1,3,
Jason A. Hanna¹^nAff7,
Catherine J. Drummond¹^nAff8,
Matthew C. Maguire¹,
Patrick J. Leavey ORCID: orcid.org/0000-0002-2822-1118¹,
David Finkelstein⁴,
Hongjian Jin⁵,
Patrick A. Schreiner⁵,
Jerold E. Rehg⁶ &
…
Mark E. Hatley ORCID: orcid.org/0000-0001-7147-3946¹

Nature Communications volume 12, Article number: 5520 (2021) Cite this article

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Abstract

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.

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Introduction

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood and high-risk RMS patients have a 5-year survival rate of only 20%¹. RMS is classified into two major histologic subgroups, alveolar (ARMS) and embryonal (ERMS)². The majority of ARMS tumors (~80%) harbor chromosomal translocations resulting in expression of either PAX3-FOXO1 or PAX7-FOXO1 oncofusion proteins which foretell a worse prognosis¹. ARMS tumors without oncofusion proteins both molecularly and clinically resemble ERMS, thus classification as either fusion-positive (FP-RMS) or fusion-negative (FN-RMS) better reflects the biological and clinical features of RMS¹. FN-RMS tumors are genetically heterogeneous with a constellation of putative driver mutations and loss-of-function alterations in tumor suppressors such as TP53 and CDKN2A; the majority of tumors have no apparent driver mutations^3,4,5,6,7. In contrast, hypermethylation of the Phosphatase and Tensin Homolog (PTEN) promoter and decreased expression was found in over 90% of FN-RMS tumors⁶. PTEN promoter hypermethylation is one of many ways that PTEN expression can be modulated in cancer^8,9. Furthermore, a subset of FN-RMS tumors also harbors PTEN mutations¹⁰.

PTEN’s tumor-suppressive function is thought to be secondary to dephosphorylation and inactivation of the lipid second messenger phosphatidylinositol-3,4,5-triphosphosphate, a potent PI3K/AKT/mTOR pathway activator^11,12,13. Inhibition of the PI3K/AKT/mTOR pathway is an attractive RMS therapy but has yielded varied results^3,14,15,16. However, increasing evidence suggests PTEN maintains a myriad of tumor-suppressive functions aside from negatively regulating the PI3K pathway¹¹. PTEN-deficient tumors require the expression of other genes to maintain their proliferative state; these synthetic essential gene interactions create therapeutically exploitable gene dependencies^17,18. Using an integrative expression and copy number computational approach, PTEN was suggested to be a key tumor suppressor in FN-RMS¹⁹. However, PTEN’s biological function in FN-RMS is largely unexplored. Herein, we leverage a Hedgehog-driven, FN-RMS mouse model to interrogate the role of PTEN in FN-RMS biology^20,21. In contrast to modulating the PI3K/AKT/mTOR pathway, we reveal PTEN loss drives a transcriptional axis centered on PAX7 necessary for FN-RMS tumor identity.

Results

Pten loss exacerbates aP2-Cre;SmoM2 FN-RMS development

The nearly universal PTEN promoter hypermethylation in FN-RMS⁶ led us to investigate whether Pten loss is sufficient to drive rhabdomyosarcomagenesis by conditionally deleting a Pten^flox allele with Cre recombinase expressed from the adipose protein 2 (aP2) promoter (Supplementary Fig. 1a)²². Neither aP2-Cre;Pten^flox/flox (AP^cKO) nor aP2-Cre;Pten^flox/+ (AP^cHet) compound mutant mice developed tumors. AP^cKO mice had significantly larger intrascapular brown adipose tissue (BAT) but not inguinal white adipose tissue (iWAT) than AP^cHet littermates (Supplementary Fig. 1b–d). Histologically, the BAT was hyperplastic without evidence of hibernoma (Supplementary Fig. 1e). The iWAT displayed hyperplastic cellularity without any overt pathology (Supplementary Fig. 1f). Thus, Pten loss is not sufficient to drive rhabdomyosarcomagenesis in aP2-Cre mice.

In order to understand the consequences of PTEN loss in FN-RMS, we sought to conditionally delete Pten^flox in our murine FN-RMS model resulting from activation of a conditional, constitutively active Smoothened allele (SmoM2) by aP2-Cre^20,21. We bred aP2-Cre;Pten^flox/+ mice to Smo^M2/M2;Pten^flox/+ mice to generate aP2-Cre;Smo^M2/+;Pten^flox/flox (ASP^cKO), aP2-Cre;Smo^M2/+;Pten^flox/+ (ASP^cHet), and aP2-Cre;Smo^M2/+;Pten^+/+ (ASP^WT) compound mutant mice (Fig. 1a). Pten loss accelerated tumor formation with a median tumor-free survival of 9 days in ASP^cKO mice compared to 16 days in ASP^WT mice (Fig. 1b and Supplementary Fig. 2a). All tumors in ASP^cKO mice occurred in the head and neck without any evidence of regional lymph node or pulmonary metastasis at necropsy just as in the ASP^WT mice. In addition, ASP^cKO mice had increased tumor burden and decreased body weight compared to ASP^WT mice (Fig. 1c and Supplementary Fig. 2b–d).

**Fig. 1: *Pten* loss exacerbates *aP2-Cre;SmoM2* FN-RMS development.**

Histologic examination revealed ASP^cKO tumors contained malignant proliferations with vague, haphazard spindle cell growth patterns comprised of cohesive cells with coalescing pale pink cytoplasm, with or without striations and irregular margins infiltrating adjacent tissue (Fig. 1d). The cells contained variably sized round, oval, or spindle nuclei. By immunohistochemistry (IHC), ASP^cKO tumors strongly expressed muscle-specific intermediate filament DESMIN and nuclear staining for skeletal muscle-specific transcription factors MYOD1 and MYOGENIN (Fig. 1d and Supplementary Fig. 2e, f). In addition, ASP^cKO tumors have a higher proliferative index than ASP^WT tumors (Fig. 1e, f). The infiltrative pattern and cytological morphology of the small cells in ASP^cKO tumors are consistent with an embryonal-like RMS rather than the well-differentiated RMS more typical of the ASP^WT tumors. Importantly, PTEN protein expression by IHC showed the absence of PTEN staining in ASP^cKO tumors, whereas both cytoplasmic and nuclear PTEN staining was observed in ASP^WT tumors (Fig. 1g). These histologic observations suggest PTEN loss may have a functional role in maintaining a less differentiated cell state.

ASP^WT tumors can be serially transplanted in NSG but not in SCID/Beige immunocompromised mice (Supplementary Fig. 2g). To test whether tumors from ASP^cKO mice are more tumorigenic than ASP^WT mice, we surgically implanted tumors into the flanks of SCID/Beige mice. All ASP^cKO tumors implanted developed tumors that were histologically similar to FN-RMS suggesting ASP^cKO tumors are more malignant than ASP^WT tumors (Fig. 1h, i). The shorter latency, increased tumor burden, and enhanced tumorigenicity illustrates ASP^cKO tumors represent a more robust model of FN-RMS.

To determine if the effects of Pten loss were not solely reflective of generic tumor suppressor loss, we conditionally deleted Trp53, Cdkn2a, and Rb1 independently in our aP2-Cre;SmoM2 model. The deletion of Trp53, Cdkn2a or Rb1 in our model did not alter the tumor latency, histology, or tumor burden despite increasing the penetrance of tumor formation (Supplementary Fig. 2h–q). While all ASP^cKO primary tumors formed secondary tumors as allografts in SCID/Beige mice, only two of six aP2-Cre;Smo^M2/+;Trp53^flox/flox and two of eight aP2-Cre;Smo^M2/+;Rb1^flox/flox formed secondary tumors while no tumors formed in aP2-Cre;Smo^M2/+;Cdkn2a^flox/flox allografts (Supplementary Fig. 2q). These data suggest that Pten loss provides a significant advantage to the RMS tumor cell.

PTEN loss enhances myogenic differentiation impairment in ASP^cKO tumors

Skeletal muscle development is a well-studied process of tissue differentiation driven by transcriptional regulation from fetal to mature adult skeletal muscle tissue²³. Both ASP^cKO and ASP^WT tumors had increased expression of early skeletal muscle transcription factors Myod1, Myf5, and Myog compared to the mature sternocleidomastoid (SCM) skeletal muscle by real-time PCR consistent with FN-RMS (Fig. 2a). Although there were differences in Myod1 and Myog mRNA expression between ASP^WT and ASP^cKO tumors, there was no change in protein expression by IHC (Fig. 1d and Supplementary Fig. 2e, f). In contrast, ASP^cKO tumors had significantly lower expression of terminally differentiated skeletal muscle genes, Acta1, Ckm, and Myh4 (Fig. 2b). This suggests that Pten loss further attenuates the differentiation of the skeletal myogenic program within the FN-RMS tumor cells.

Similar to the series of transcription factors driving skeletal muscle determination and differentiation, myosin heavy chain (MyHC) isoforms of sarcomeric filaments display a developmental progression from MyHC-embryonic (MYH3) to MyHC-neonatal (MYH8) to finally adult MyHCs (MYH1, MYH2, MYH4)²³. MyHC-embryonic encoded by Myh3 which is only expressed in embryonic developing skeletal muscle is also increased in the ASP^cKO tumors compared to ASP^WT. However, no change in expression with neonatally expressed Myh8 suggests ASP^cKO tumors resemble an earlier skeletal muscle progenitor (Fig. 2c). To better define the tumor cell differentiation state in the ASP^cKO and ASP^WT tumors, we bred Rosa26^tdTomato reporter mice into the ASP^WT and ASP^cKO models to generate ASTP mice. In the ASTP mice, tumor cells that have expressed aP2-Cre are indelibly labeled with tdTomato allowing tumor cell visualization. Immunofluorescent staining illustrated that nearly all tdTomato⁺ tumor cells in ASTP^WT mice expressed adult MyHC compared to only ~60% of the tdTomato⁺ tumor cells in ASTP^cKO mice (Fig. 2d, e). Furthermore, triple the number of tdTomato⁺ tumor cells in ASP^cKO mice were positive for MyHC-embryonic compared to ASP^WT mice while no change was observed for MyHC-perinatal (Fig. 2f, g and Supplementary Fig. 3a, b). These results further indicate that ASP^cKO tumors are less differentiated than ASP^WT tumors and more reflective of human ERMS.

PTEN loss activates AKT but not mTORC1 in ASP^cKO tumors

Next, we sought to determine how PTEN loss affects cell signaling in the ASP^cKO tumors. PTEN loss and unchecked PI3K-pathway activation lead to aberrant activation/phosphorylation of kinases important in maintaining many hallmarks of cancer¹¹. To determine the signaling pathways regulated by PTEN loss in ASP^cKO tumors, we performed an unbiased analysis of signaling proteins using reverse-phase protein arrays with 350 different antibodies directed against major cancer-related proteins. In ASP^cKO tumors, 30 probes were relatively overexpressed and 25 probes were relatively underexpressed (Fig. 3a). As expected, PTEN was the protein most decreased in ASP^cKO compared to ASP^WT tumors with concomitant increased AKT phosphorylation at both Thr308 and Ser473 (Fig. 3a, b). Among other proteins within the AKT/mTOR pathway, only phosphorylated PRAS40^Thr246 was significantly relatively overexpressed in the ASP^cKO tumors. Surprisingly, the mTOR-downstream target phosphorylated p70S6K^Thr389 was significantly decreased in ASP^cKO tumors, suggesting that AKT phosphorylation was largely uncoupled from mTORC1, further evidenced by lack of increased phosphorylation of S6 or 4E-BP1 (Fig. 3a, b). The other proteins increased in ASP^cKO tumors included both NOTCH1 and NOTCH3, constituents of MAPK cascades, and phosphorylated ERBB3 with its cognate ligand HEREGULIN. We confirmed increased ERK1/2 phosphorylation in ASP^cKO bulk tumor lysates (Supplementary Fig. 4a). However, we saw no increased sensitivity to trametinib in ASP^cKO primary rhabdospheres; this was probably due to a lack of maintained ERK1/2 phosphorylation observed in cultured ASP^cKO primary rhabdospheres (Supplementary Fig. 4b, c). Furthermore, immunosuppressive molecules B7-H3, CD86, and CD38 as well as necroptosome components cleaved caspase-8 and RIP3 (also known as RIPK3) were relatively underexpressed in ASP^cKO tumors suggesting less immune surveillance in the tumor microenvironment with Pten loss.

Given the increased AKT phosphorylation and absence of concomitant increased mTOR signaling in the ASP^cKO tumors, we examined whether these signaling results correlated with increased sensitivity to PI3K, mTOR, or AKT inhibitors. We dissected and dissociated ASP^WT and ASP^cKO tumors, seeded dissociated tumor cells to grow as rhabdospheres, and assayed cell growth when incubated with either GDC-0941 (pan-PI3K inhibitor), MK-2206 (AKT inhibitor), everolimus (mTOR inhibitor), or doxorubicin^20,24. ASP^cKO rhabdospheres displayed increased sensitivity to GDC-0941 but no difference in sensitivity to MK-2206, everolimus, or doxorubicin (Fig. 3c–f). Although ASP^cKO tumors show increased phosphorylation of AKT, these results suggest the phenotypic differences observed in ASP^cKO tumors are most likely mediated through PI3K or alternative PTEN functions.

Dbx1 is the most overexpressed gene in ASP^cKO tumors and has a functional role in human FN-RMS

Given PTEN nuclear localization in ASP^WT tumors, we sought to determine if known nuclear functions were responsible for the ASP^cKO phenotype. Neither DNA damage nor chromosome instability was increased in the ASP^cKO tumors (Supplementary Fig. 5a–c). We next explored whether PTEN regulated gene expression and analyzed the gene expression changes in bulk ASP^WT and ASP^cKO tumors as well as SCM muscle. Principal component analysis revealed that gene expression in ASP^WT and ASP^cKO tumors clustered distinctly; however, they were more similar to each other than to mature SCM skeletal muscle (Fig. 3g and Supplementary Data 1). Comparing gene expression in ASP^cKO to ASP^WT tumors, 248 genes had increased and 286 genes had decreased expression (≥twofold, P value <0.05) (Fig. 3h). Several differentially expressed genes were validated by real-time PCR (Fig. 3i). Gene ontology analysis of the 286 genes with decreased expression in ASP^cKO tumors compared to ASP^WT tumors uncovered gene ontology (GO) terms associated with skeletal myogenic differentiation consistent with increased expression of embryonic MyHC and decreased expression of adult MyHC in ASP^cKO tumors, and dysregulated immune responses (Figs. 2d–g and 3j); conversely, 248 genes with increased expression (Supplementary Data 1) revealed GO terms associated with increased proliferation and DNA replication consistent with increased Ki67 staining in ASP^cKO tumors (Figs. 1e, f and 3k). Furthermore, we utilized CIBERSORT to analyze gene expression profiles from ASP^WT and ASP^cKO tumors to predict immune cell composition; ASP^cKO tumors were predicted to have less naive B cells, M1 macrophages, resting mast cells, and eosinophils while M0 macrophages are enriched in the ASP^cKO tumors (Supplementary Fig. 4d).

The subset of genes relatively overexpressed in ASP^cKO tumors included neuronal transcription factors including Dlx5 and Pax7 as well as Dbx1 (Developing brain homeobox 1), the most overexpressed gene in the ASP^cKO tumors (Fig. 4a). DBX1 is a homeobox transcription factor thought to function as a transcriptional repressor critical in neural patterning of innate processes^25,26,27. However, the role of DBX1 in cancer is unknown.

**Fig. 4: *Dbx1* is the most overexpressed gene in ASP^cKO tumors and has a functional role in human FN-RMS.**

Given the lack of knowledge about DBX1’s function in cancer, we first interrogated the Cancer Dependency Map²⁸ and found that DBX1 highly expressed in RMS cell lines with only rhabdoid tumors exhibiting higher expression (Fig. 4b). Next, we probed two public human RMS expression datasets both illustrating increased DBX1 expression in FN-RMS (Fig. 4c and Supplementary Fig. 6a)^29,30. Furthermore, increased DBX1 expression was also seen in all FP-RMS and most FN-RMS patient-derived xenografts (PDX) from the Childhood Solid Tumor Network at St. Jude compared to primary human myoblasts (Fig. 4d)³¹. Therefore, DBX1 expression is increased in both mouse and human FN-RMS; however, the functional significance of DBX1 in RMS biology remains unknown.

To study the role of DBX1 in human FN-RMS, we generated a cell line from FN-RMS PDX SJRHB015721_X1 with high DBX1 expression (Fig. 4d and Supplementary Fig. 6b, c). We transduced the cells with lentivirus expressing either two independent shRNAs targeting DBX1 or a scramble shRNA control. DBX1-targeting shRNAs decreased DBX1 expression and cell viability compared to the scramble control, suggesting a role for DBX1 in FN-RMS (Fig. 4e, f). To further understand the functional role of DBX1 in RMS, we performed gene expression analysis on the SJRHB015721_X1 cell line transduced with the two shRNAs and scramble control. 197 genes had decreased expression and 156 genes had increased expression in both shRNAs compared to scramble control (absolute log fold change >0.58, −log₁₀ adjusted P < 0.5) (Fig. 4g and Supplementary Fig. 6d and Supplementary Data 2). Gene ontology analysis of the 197 genes with decreased expression with DBX1 knockdown highlights processes related to cell proliferation (Fig. 4h and Supplementary Data 2). The GO terms of the 156 genes with increased expression upon DBX1 knockdown were enriched for apoptotic processes as well as positive regulators of development and morphogenesis (Fig. 4i and Supplementary Data 2). These data suggest the DBX1 corepressor activity assists in maintaining the proliferative state and blocked differentiation in FN-RMS.

To determine the in vivo significance of DBX1, we dissociated two human FN-RMS PDX samples, SJRHB015721_X1 and SJRHB012405_X1, and transduced cells with lentiviral vectors expressing EGFP as a marker of transduction and shRNA to knockdown DBX1 expression. After transduction, cells were injected into the gastrocnemius of SCID/Beige mice and used to establish primary rhabdosphere cultures. DBX1 expression decreased with shRNA expression in rhabdospheres indicating efficient DBX1 knockdown after viral transduction (Supplementary Fig. 6e, f). DBX1 knockdown in both SJRHB015721_X1 and SJRHB012405_X1 PDXs reduced tumor volume and tumor mass (Fig. 4j, k and Supplementary Fig. 6g, h, j, k). Gross and immunohistochemical analysis indicated that the DBX1 knockdown tumors were indistinguishable from the scramble controls except that the shDBX1 tumors that formed lost EGFP expression (Fig. 4l and Supplementary Fig. 6i, l). This suggests that tumors developing from shDBX1-transduced cells had escaped lentiviral transduction and thus escaped DBX1 knockdown highlighting the importance of DBX1 in FN-RMS.

PAX7 controls Dbx1 expression

Given Dbx1 is the most overexpressed gene in ASP^cKO tumors and DBX1 knockdown decreased tumor growth in human FN-RMS cells, we sought to determine how Pten loss regulates Dbx1 expression. We first examined a 1 kb promoter region immediately 5’ of Dbx1’s initiating ATG to identify known transcription factor binding motifs and compared this to genes with >twofold expression in ASP^cKO tumors and found only one overlapping gene – Pax7 (Fig. 5a). PAX7 is a transcription factor critical in both adult muscle stem cell (satellite cells) maintenance and preserving a dedifferentiated state in FN-RMS^32,33. Interestingly, PAX7 along with MYOD1 are the only genes that are both members of FN-RMS core-regulatory circuits and are FN-RMS dependencies thus coupling tumor cell identity and survival (Fig. 5b)^34,35. We confirmed increased Pax7 expression in ASP^cKO tumors compared to ASP^WT tumors by real-time PCR, immunoblotting, and immunofluorescence (Fig. 5c–f). To confirm the link between PTEN and Pax7 transcription, we infected Pten^flox mouse embryonic fibroblasts (MEFs) with adenovirus encoding either Cre recombinase or GFP. Adeno-Cre-infected Pten^flox MEFs exhibited decreased Pten expression and increased Pax7 (Fig. 5g). Next, to determine if PAX7 transcriptionally regulates Dbx1, we cloned 900 bp of the Dbx1 promoter containing a putative PAX7-binding site upstream of luciferase. PAX7 expression led to a dose-dependent increase in Dbx1-promoter-driven luciferase activity (Fig. 5h, i). Next, we transduced wild-type MEFs with lentivirus overexpressing PAX7 and showed concomitant increased Dbx1 expression (Fig. 5j). Conversely, shRNA knockdown of Pax7 in ASP^cKO rhabdospheres resulted in decreased Dbx1 expression (Fig. 5k). Furthermore, decreased PAX7 expression in human rhabdospheres led to decreased DBX1 expression (Fig. 5l).

The murine and human DBX1 proximal promoters are highly conserved (Fig. 5m). Of the two putative PAX7-binding sites within the human DBX1 promoter, only the proximal (site 1) is conserved in the mouse. We designed primers that flank each of these human sites and performed chromatin immunoprecipitation (ChIP)-qPCR to analyze whether PAX7 is bound to the DBX1 promoter. PAX7 binding was enriched eightfold over control IgG at the proximal DBX1 promoter PAX7-binding motif (which also corresponds to the mouse Dbx1 promoter) (Fig. 5n). These data indicate PTEN loss increases PAX7 expression and DBX1 is a downstream target of PAX7 in FN-RMS.

Human FN-RMS is dependent on PAX7

Given that PAX7 is putatively both a transcription factor in the RMS core-regulatory circuit and a dependency in RMS, we sought to further explore the role of PAX7 in RMS. The Broad Institute’s Cancer Dependency Map (DepMap) illustrates that PAX7 is highly expressed in soft-tissue sarcoma cell lines (Fig. 6a)²⁸. Specifically, PAX7 expression is increased in human FN-RMS tumors compared to skeletal muscle in a public human RMS expression dataset (Fig. 6b)³⁰, as well as in human FN-RMS cell lines and PDXs compared to myoblasts (Supplementary Fig. 7a, b). CRISPR/Cas9-dependency screening in DepMap revealed a PAX7 dependency in FN-RMS cell lines (Fig. 6c). Thus, we wanted to confirm if human FN-RMS cell lines and PDXs are dependent on PAX7 expression. Using lentiviruses expressing two independent shRNA targeting PAX7 or a scramble control, we performed PAX7 knockdown in human FN-RMS cell lines (SMS-CTR, RD, Rh18) and cell lines derived from two PDXs – SJRHB015721_X1 and SJRHB011_Y (Fig. 6d and Supplementary Fig. 7c, d). PAX7 knockdown in FN-RMS cells decreased both cell viability and cell growth in vitro suggesting a role in FN-RMS (Fig. 6e, f and Supplementary Fig. 7e–h).

Since PAX7 knockdown reduced FN-RMS cell proliferation in vitro, we aimed to determine whether shPAX7 knockdown affected tumorigenicity in vivo. SMS-CTR cells transduced with shPAX7 or scramble control were injected into the gastrocnemius of SCID/Beige mice. PAX7 knockdown significantly reduced tumor volume and mass in SMS-CTR cell xenografted mice (Fig. 6g, h, i). To establish this was not a consequence or artifact of using an established cell line, we extended our analysis to FN-RMS PDXs. SJRHB015721_X1 and SJRHB012405_X1 PDX cells were dispersed into single cells and transduced with lentivirus expressing shPAX7 or scramble control. After a brief incubation at 37 °C, the cells were either placed in spheroid culture or injected into the gastrocnemius muscle of SCID/Beige mice. PAX7 knockdown was confirmed in spheroid culture cells (Supplementary Fig. 7i). In both SJRHB015721_X1 and SJRHB012405_X1 PDXs, PAX7 knockdown with shPAX7 resulted in decreased tumor volume (Fig. 6j and Supplementary Fig. 7j); however, tumor mass only decreased in SJRHB015721_X1 (Fig. 6k and Supplementary Fig. 7k). Tumors from SJRHB012405_X1 retained PAX7 expression at the endpoint suggesting a negative selection for cells with PAX7 knockdown (Fig. 6l and Supplementary Fig. 7l). Together, our in vitro and in vivo data confirm PAX7 is a dependency in FN-RMS cells.

PAX7 dictates rhabdomyosarcoma tumor cell identity in ASP^cKO tumors

To elucidate the mechanistic role of PAX7 in FN-RMS, we returned to our ASP^cKO mouse model. We bred Pax7^flox alleles into the ASP^cKO model to generate compound conditional knockout mice, aP2-Cre;Smo^M2/+;Pten^flox/floxPax7^flox/flox (ASP^cKOP7^cKO) and associated littermate control mice (Fig. 7a). Deleting Pax7 in ASP^cKO mice rescued the decreased tumor-free survival and increased tumor mass associated with Pten loss in the AS mice (Fig. 7b, c). ASP^cKOP7^cKO tumors were noncircumscribed and highly infiltrative with the tumor cells perpendicular to and through the skeletal muscle fibers (Fig. 7d). Histologically, the ASP^cKOP7^cKO tumor cells stained sporadically for DESMIN but were no longer positive for RMS diagnostic markers MYOD1 and MYOGENIN; this was specific for ASP^cKOP7^cKO tumors as ASP^cHetP7^cKO and ASP^cKOP7^cHet tumors were still myogenic, small round blue tumors consistent with RMS (Fig. 7d). The ASP^cKOP7^cKO tumors were also significantly less proliferative than either ASP^WT and ASP^cKO tumors (Fig. 7e). However, these phenotypes cannot be attributed to differences in PI3K-pathway activation observed in ASP^cKO and ASP^WT tumors as ASP^cKOP7^cKO tumors had similar AKT and S6 phosphorylation as ASP^cKO tumors (Fig. 7f). This highlights a unique link between PTEN and PAX7 independent of PI3K-pathway signaling that illustrates PAX7 is a synthetically essential gene in PTEN-deficient FN-RMS.

**Fig. 7: PAX7 loss rescues deleterious effects of *Pten* loss in ASP^cKO tumors.**

To determine the tumor type produced following concomitant Pten and Pax7 depletion, we first performed ultrastructural analysis and that revealed ASP^cKOP7^cKO tumor features were no longer consistent with skeletal muscle but with leiomyosarcoma (LMS), including elongated cellular shape, grooved nuclei, and prominent rough endoplasmic reticulum (Fig. 8a). Intriguingly, ASP^cKOP7^cKO tumors but not ASP^WT or ASP^cKO tumors expressed the LMS markers smooth-muscle actin (SMA) and CALDESMON (Fig. 8b). Given that ASP^cKOP7^cKO tumors had a lower proliferative index (Fig. 7e) and did not engraft into SCID/Beige immunocompromised mice (0/6 tumors), the tumors that arise may lie along a spectrum of low-grade sarcomas with smooth-muscle immunophenotypic features. These results illustrate that the Pten loss phenotype is dependent on PAX7 and indicates that PAX7 controls FN-RMS cell fate.

**Fig. 8: PAX7 dictates rhabdomyosarcoma identity in ASP^cKO tumors.**

To explore the mechanisms altering ASP^cKOP7^cKO tumor identity, we compared the gene expression in ASP^cKO and ASP^cKOP7^cKO tumors. We bred the Rosa26^tdTomato reporter mouse into our ASP^cKOP7^cKO model to generate ASTP^cKOP7^cKO compound mutant mice allowing us to use flow cytometry to purify tumor cells by sorting tdTomato-positive and CD31 (PECAM)-negative (to remove endothelial cells) cells (Supplementary Fig. 8a)²⁰. RNAseq from sorted Tom⁺CD31⁻ tumor populations revealed robust differential gene expression with 973 genes enriched in ASTP^cKO and 1002 genes enriched in ASTP^cKOP7^cKO tumor cells (≥twofold, P < 0.05) (Fig. 8c, d and Supplementary Data 3). Functional annotation clustering and gene set enrichment analyses further highlighted differences between ASTP^cKO and ASTP^cKOP7^cKO tumors with myogenic and neurogenic transcriptional programs enriched in ASTP^cKO and relatively underexpressed in ASTP^cKOP7^cKO (Fig. 8e, f, h, i and Supplementary Data 3). This dual regulation of skeletal myogenic and neurogenic genes is important to note as PAX7 is a transcription factor that regulates both developmental programs^36,37. Curiously, all five distal-less homeobox genes were also relatively underexpressed in ASTP^cKOP7^cKO, indicating PAX7 as a master regulator of their expression (Fig. 8e).

In contrast to the increase in skeletal myogenic and neurogenic transcriptional programs enriched in the ASTP^cKO tumors, there were relatively few transcriptional programs increased in ASTP^cKOP7^cKO tumors (Fig. 8e, g, h, j and Supplementary Data 3). The major pathways enriched in ASTP^cKOP7^cKO tumors included calcium signaling, cyclic nucleotide pathways, and most intriguingly, vascular smooth muscle including the smooth muscle master-regulator transcription factor Myocardin (Myocd)³⁸. Furthermore, ASTP^cKOP7^cKO tumors were enriched for genes associated with LMS, including smooth-muscle structural proteins Acta2 and Myh11. Most of the LMS genes (Lmod1, Casq2, Actg2, Mylk, and Slmap) are markers of subtype I of human LMS which is mostly found in extrauterine sites³⁹. Unlike the expression differences in Lmod1, Myh11, and Acta2 observed between ASP^cKOP7^cKO and ASP^cKO tumors (Supplementary Fig. 8b), we did not find any comparable increases in LMOD1, MYH11, and ACTA2 following shRNA-mediated knockdown of PAX7 expression in three human FN-RMS cell lines/PDXs—SMS-CTR, SJRHB015721_X1, and SJRHB012405_X1—highlighting that this PAX7-controlled fate decision occurs early in tumorigenesis (Supplementary Fig. 8c–e). Furthermore, tumors that form in xenografts of SMS-CTR cell line and the SJRHB015721_X1 and SJRHB12405_X1 PDXs following PAX7 knockdown have not shifted fate to LMS by IHC for SMA and CALDESMON (Supplementary Fig. 8f). These results indicate that deletion of Pax7 in the ASP^cKO murine FN-RMS model yields tumors whose histology, ultrastructure, and gene expression recapitulates low-grade sarcoma with smooth-muscle differentiation and not a skeletal myogenic tumor-like FN-RMS. Therefore, simultaneous Pten and Pax7 loss in murine FN-RMS not only rescued the effects of Pten loss in these tumors but identified a synthetic essential relationship between Pten and Pax7 controlling tumor cell fate.

Discussion

The cell of origin and genetic perturbations cooperate in tumor cell transformation and fate determination highlighting cellular and developmental pliancy in tumorigenesis. Genetically engineered mouse models offer a method to dissect these relationships by utilizing aP2-Cre to activate oncogene and/or delete tumor suppressor expression. Genetic fate mapping reveals aP2-Cre expression in white and brown adipocytes, dorsal root ganglia, macrophages, and endothelial cells^20,22,40. Compound mutant mice using aP2-Cre to drive recombination resulting in biallelic Dicer1 deletion or the combination of oncogenic Kras^G12D activation and Cdkn2a deletion result in angiosarcoma from the transformation of endothelial cells^20,41. However, activation of constitutively active, oncogenic SMO^M2, and hedgehog pathway signaling by aP2-Cre results in cell reprogramming and transformation of endothelial progenitor cells driving FN-RMS tumorigenesis²⁰. We now find Pten loss cooperates with SMO^M2 in aP2-Cre expressing cells to drive a less differentiated tumor that better recapitulates the most common human embryonal RMS histology. This was specific to Pten loss as Cdkn2a, Rb1, or Trp53 loss did not phenocopy the results. The effects of Pten deletion were dependent on PAX7 as Pax7 deletion rescued the phenotypes of Pten loss and altered tumor cell fate resulting in a spectrum of low-grade sarcomas with immunophenotypic smooth-muscle differentiation, including LMS. Interconnections between RMS and LMS have been described with anecdotal clinical reports of LMS co-occurring with RMS or with rhabdomyosarcomatous-like lesions^42,43. This suggests there may be some shared developmental trajectories and highlights the fluidity of cellular and developmental pliancy in tumorigenesis.

Dbx1 was the most relatively overexpressed gene in ASP^cKO tumors and has not been reported to have a role in cancer. DBX1 is an evolutionarily conserved homeobox transcription factor expressed embryonically in central nervous tissue functioning as a transcriptional corepressor to establish specific neuron fate and neural patterning^25,26,27. We found that both human FN-RMS and FP-RMS had subsets of tumor with high DBX1 expression. Interestingly, DBX1 transcription was controlled by the skeletal muscle satellite cell marker PAX7 in both murine and human FN-RMS models. Since FP-RMS tumors are driven by PAX3-FOXO1 or PAX7-FOXO1 fusion oncoproteins containing either PAX3 or PAX7 DNA binding domains, we postulate that DBX1 expression is driven transcriptionally by the fusion oncoproteins much how PAX7 itself targets DBX1 expression in FN-RMS. This may suggest some shared etiology between two major RMS subtypes.

Importantly, PAX7 not only functions in skeletal muscle development and maintenance of satellite cell quiescence but also other cell types, including pituitary gland melanotropes, neural tube, and actively migrating neural crest cells^36,37,44,45. ASP^cKO tumors engage a transcriptional program composed of several neural-associated genes, many of which are under the control of PAX7 as their expression is also lost in ASP^cKOP7^cKO tumors. Furthermore, neural stem cells have the ability to convert to a myogenic lineage and the myogenic determination factor MYOD1 can bind to neural-specific genes^46,47. It has also been shown that Pten loss drives Pax7 mRNA increases in human neural stem cells further highlighting how transcriptional regulation by PTEN may be a critical mediator of its tumor suppressor functions⁴⁸. This may provide therapeutic vulnerability as it appears that FN-RMS cells are dependent on these lineage factors^35,49.

Engaging the PAX7 transcriptional program, such as with DBX1, in these FN-RMS tumors by Pten loss highlights the necessity in understanding the complete physiological consequences of PTEN loss in tumors. This includes potential changes in the immune microenvironment as our RPPA data suggest a more immunosuppressive environment, much like other tumors with PTEN loss^50,51,52. Furthermore, increasing evidence indicates that PTEN mediates its tumor-suppressive effects via not only PI3K-pathway-dependent mechanisms but also through a myriad of PI3K-pathway-independent mechanisms^11,53. PTEN-deficient cancers are known to have synthetic essential relationships with CHD1 and PDHK1^17,18. Concomitant Pax7 and Pten loss completely rescued the deleterious effects of the latter in our FN-RMS model suggesting Pten loss contributes to rhabdomyosarcomagenesis in a PI3K-pathway-independent mechanism via a synthetic essential interaction. It remains unclear how pervasive these synthetic essential relationships between tumor suppressor and lineage factors are in FN-RMS or other tumors. PAX7 is only one component of the FN-RMS core-regulatory circuits (CRCs)^34,35. Do other components of the FN-RMS CRC phenocopy this result? Furthermore, other CRCs in additional tumor types are becoming clearer; do similar mechanisms underlying genetic alteration context, cell type, and transcriptional states give rise to lineage determinants and dependencies? These are open questions that need to be empirically explored.

The combination of near-universal PTEN perturbation in human FN-RMS and PI3K-pathway-independent mechanisms in maintaining tumor identity in murine FN-RMS indicates PTEN’s role in activating RMS-specific transcriptional outputs. This work intertwines the interplay between context-dependent tumor suppressor loss and transcriptional activation can engage a synthetic essential pairing; here, the combination of PTEN loss and PAX7 activation not only maintains tumor growth and survival but maintains tumor identity. Here, we show that the ASP^cKOP7^cKO tumors are no longer FN-RMS. The concomitant loss of PTEN and PAX7 gave rise to smooth-muscle-like tumors that lie along a spectrum of smooth myogenic differentiation from benign smooth-muscle-like tumors to those that appear like leiomyosarcoma. It is possible that the dual PTEN/PAX7 loss in our Smoothened-driven tumors allows the outgrowth of a precursor lesion to become more fully manifested. Furthermore, since PAX7 depletion in established tumors did not cause a shift to smooth-muscle differentiation, our data suggest that these fate decisions are made early on in tumorigenesis and that tumor lineage factors like PAX7 become more focused on tumor maintenance after these fate decisions are made. This may explain why development-related tumors, such as pediatric tumors like RMS, are highly dependent on transcriptional regulators⁵⁴. Although their loss early on in tumorigenesis may alter the ultimate tumor presentation, clinically, these tumors may have become more addicted to these factors in tumor maintenance; this may allow for therapeutic intervention of these tumors—either through indirect mechanisms (such as with HDAC inhibition in FP-RMS) or with directly-targeted molecular glues or protein degraders as these technologies improve.

The presented work details how the synthetic essential relationship dictates tumor fate in vivo. This synthetic essential relationship between Pten and Pax7 highlights the complex interplay between genetic alterations and transcriptional states that not only define tumorigenicity but even tumor origin and evolution. Taken together, this work illustrates how incredibly complex the interactions between the cell of origin, mutational spectrum, and transcriptional state are in dictating tumor lineage, and ultimately, therapeutic vulnerabilities.

Methods

Genetically engineered mouse models

All mouse strains are reported: aP2-Cre²², R26-tdTomato (#7914, The Jackson Laboratory (JAX))⁵⁵, SmoM2 (#5130, JAX)⁵⁶, Pten^flox (#6440, JAX)⁵⁷, Pax7^flox (#12653, JAX)⁵⁸, Cdkn2a^flox (Nabeel Bardeesy⁵⁹, Trp53^flox (#8462, JAX)⁶⁰, and Rb1^flox (#01XC1, US NCI)⁶⁰. All mice were fed and watered ad libitum with consistent access to food and water, and all mice were housed in a facility kept at ambient temperature and humidity with 12-h light/12-h dark cycles. Anterior necks lightly palpated while scruffed to feel for tumors and scored when first felt. All animal experiments were reviewed and approved by the SJCRH Institutional Animal Care and Use Committee.

Cell lines, primary cell culture, and patient-derived xenografts (PDXs)

293T (Martine Roussel, SJCRH), RD (#CCL-136, ATCC), Rh18 (Children’s Oncology Group Cell Line Repository, Monrovia, CA, USA), SMS-CTR (Rene Galindo, UTSW Medical Center), Rh36 (Christopher Morton, SJCRH), TE441 (#CRL-7677, ATCC), Rh30 (#CRL-2261, ATCC), and Rh2, Rh6, Rh3, Rh4, Rh28, and Rh41 (Gerard Grosveld, SJCRH) cells were maintained in DMEM (#SH32043, HyClone) supplemented with 10% fetal bovine serum (Hyclone) and 1% antibiotic/antimycotic (#A5955, Sigma). SJRHB015721_X1 and SJRHB011_Y PDX-derived cell lines were harvested from SCID/Beige hindlimbs, dissociated, and plated in complete neurobasal media as described^20,24. Briefly, tumors were digested by mechanical and enzymatic digestion with a buffered solution of collagenase II and trypsin for 1 h at 37 °C. Digested tumors were then filtered into single-cell suspensions and then plated into tissue-culture-treated plates. Primary rhabdospheres from ASP^cKO tumors and PDXs were dissociated similarly as above, except in the case of the primary mouse tumors the enzymatic digestion buffer contained collagenase B and dispase II; tumor or PDX cells were plated in low-attachment plates after sterile filtering into single-cell suspensions. Cell lines authenticated by short tandem-repeat profiling and monitored for mycoplasma contamination (Universal Mycoplasma Detection Kit, ATCC, #30-1012 K). Human skeletal muscle myoblasts obtained from Lonza (#CC-2580) and grown according to the manufacturer’s instructions. Mouse embryonic fibroblasts (MEFs) were harvested from E11.5 to 13.5 embryos. Wild-type MEFs immortalized by retroviral transduction with large T (genomic) antigen (#1778, Addgene, a gift from Bob Weinberg⁶¹). All cell lines and rhabdospheres were maintained in a humidified incubator at constant 37 °C and 5% CO₂.

RMS PDX samples (SJRHB010468_X1, SJRHB010463_X16, SJRHB010927_X1, SJRHB010928_X1, SJRHB013757_X2, SJRHB012405_X1, SJRHB013758_X1, SJRHB013759_X1, SJRHB013758_X2, SJRHB015721_X1, SJRHB15720_X1, SJRHB011_X, SJRHB011_Y, SJRHB012_X, SJRHB012_Y, SJRHB012_Z, SJRHB013_X, and SJRHB000026_X1) were obtained from the Children’s Solid Tumor Network (CSTN)³¹. The CSTN serves as a repository for deidentified PDXs developed under the MAST (Molecular Analysis of Solid Tumors) protocol from NCT01050296⁶². NCT01050296 was approved by St. Jude Children’s Research Hospital’s Institutional Review Board. Informed consent was obtained from each trial participant prior to be enrolled in the MAST clinical trial.

Histology and immunostaining

Fresh frozen or formalin-fixed paraffin-embedded tumor tissues were prepped for immunohistochemistry as described²⁰. Briefly, paraffin-embedded tissues were rehydrated followed by antigen-retrieval (conditions listed in Supplementary Table 1). Frozen tissue sections were fixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose, 2 mM MgCl₂. Fixed tumor tissue was flash-frozen in liquid-nitrogen-cooled 2-methyl-butane and sectioned on a conventional cryostat. Hematoxylin & eosin (H&E) plus immunohistochemistry were performed using standard staining procedures. Antibodies are listed in Supplementary Table 2. Images captured on a Nikon Eclipse 80i upright fluorescent microscope or a Leica DMi 8 Thunder Imager inverted fluorescent microscope; images analyzed with either Nikon Elements Basic Research (v4.1.3) or the Leica Application Suite X v3.7.2 software. For PAX7 immunofluorescence, signal amplification with the TSA-Plus Cyanine 5 (#NEL745001KT, PerkinElmer) performed according to the manufacturer’s instructions. Images quantified with Nikon Elements Basic Research (v4.1.3) or FIJI’s (ImageJ, NIH) total nuclei cell counter. Specimens for transmission electron micrographs prepared and imaged as described in⁴¹. Briefly, tumor tissue was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde, in 0.1 M sodium cacodylate buffer (pH 7.4) and post-fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer with 0.15% potassium ferrocyanide. Samples were dehydrated in increasing ethanol concentration washes to propylene oxide. Samples embedded overnight at 70 °C in epoxy resin; ultrathin (80 nm) sections were imaged on a JEOL 1200EX TEM with an AMT XR111 or Tecnai TF20 TEM with an AMT XR41 camera.

Fluorescence-activated cell sorting

ASP^cKO and ASP^cKOP7^cKO tumors dissected and manually dissociated, digested, and stained for FACS prior to analysis with a FACS Aria Cellsorter (BD Biosciences) as described²⁰. Briefly, tumors were mechanically and enzymatically digested in a buffered solution of collagenase B and dispase II for 1 h at 37 °C; tumor cells were then sterile filtered to single-cell suspensions and stained for 30 min with 1:50 anti-CD31-APC prior to flow cytometry. Antibody information is detailed in Supplementary Table 2. DAPI was used for live/dead cell marker.

Viability assays and pharmacological agents

Cells were assessed for viability with CellTiterGlo (#G7570, Promega) or periodic image acquisition with confluence calculation (Incucyte S3, Essen BioScience). CellTiterGlo assays were performed according to the manufacturer’s protocol; luminescence was measured on a BioTek Synergy 2 and BioTek’s Gen5 1.11 software. Doxorubicin (#D-4000) and everolimus (#E-4040) were from LC Labs. GDC-0941 (#S1065), trametinib (#S2673) and MK-2206 (#S1078) were from Selleck Chemicals.

Molecular cloning, viral transduction, and luciferase assays

Gene blocks (Integrated DNA Technologies (IDT), Coralville, IA, USA) designed for the murine Pax7 mRNA coding sequence were cloned into the pBABE-puro retroviral vector (Hartmut Land, Jay Morgenstern, and Bob Weinberg, #1764, Addgene). Retroviruses packaged with pCL-Eco (Inder Varma, #12371, Addgene) and transfected into 293T cells. MEFs were transduced with conditioned 293T media containing viral particles after filtering (0.22 μm)⁶³.

Lentiviruses encoding shRNAs (pLKO.1 or pLKO.3 G vectors, Bob Weinberg, #8453, Addgene⁶⁴, or Christophe Benoist and Diane Mathis, #14748, Addgene) packaged and transfected into 293T cells, and adherent cells were transduced as the MEFs above⁶⁵. For primary murine rhabdosphere transduction, virus was filtered and incubated overnight at 4°C in one-third volume of Lenti-X (#631232, Takara). Concentrated viral particles in 1–2 mL of complete neurobasal medium and plated in ultra-low-attachment plates for overnight transduction at 37 °C. For primary human PDX transduction, the filtered virus was added to pelleted PDX cells and incubated for 2.5 h at 37 °C.

MEFs infected with adenovirus-Cre recombinase (VVC-U of Iowa-1174) or GFP (VVC-U of Iowa-4) (University of Iowa Viral Vector Core) overnight (1 μg virus/1 mL medium).

The 900 bp cis-regulatory region immediately prior to the Dbx1 transcription start site was synthesized (gene block, IDT) and subcloned into pGL4.25[luc2CP/minP] (#E8431, Promega). Synthesized Pax7 cDNA was cloned into pCMV6 (Eric Olson). 293Ts were co-transfected with 280 ng pGL4.25-Dbx1 promoter, 20 ng pCMV6-LacZ (as transfection control), and dose-dependent increases in Pax7 cDNA. FuGene (#E2692, Promega) was transfection reagent. Luciferase activity was measured using the Promega Luciferase Assay System (Promega, E1501), and fluorescence activity was measured using the LacZ/Galactosidase Quantification Kit (FluoReporter, ThermoScientific, F-2905); both measurements were made on a BioTek Synergy 2. Luciferase activity was normalized to β-galactosidase activity relative to control⁶⁵.

Chromatin immunoprecipitation

Low-passage SJRHB015721_X1-adherent cells were grown to ~80% confluency; cells were collected in PBS by scraping. Cells were cross-linked in 1.1% formaldehyde in PBS, quenched with 125 mM glycine, and lysed in ChIP lysis buffer 3 [10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine] with 1× protease inhibitors, 1 mM DTT, and 1 mM PMSF. The fixed and lysed cells were sonicated with a Covaris M220 (Covaris) sonicator with the following conditions: peak incident power—75, duty factor—15%, cycles per burst—200, temperature—7 °C for 12 min in 1 mL lysis buffer. Chromatin was incubated with pre-washed protein A/G Dynabeads (Life Technologies) and 4 μg PAX7 antibody (DSHB) overnight at 4 °C. After incubation, beads were washed and immunoprecipitates were eluted. DNA from eluates was isolated using standard phenol:chloroform extraction and resuspended in low-EDTA TE buffer. Supplementary Table 1 lists the ChIP-qPCR primers used. ChIP-qPCR signals were calculated as fold enrichment over IgG control antibody.

Spectral karyotyping

ASP^cKO or ASP^WT tumors were analyzed for genomic alterations. Mice were intraperitoneally injected with 400 μL of 10 μg/mL colcemid and tumors were harvested 4 h post injection. Tumor cells were cultured in RPMI-1640 (supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) and harvested up to 10 days post culture using routine cytogenetic methods. A commercially prepared SKY probe from Applied Spectral Imaging (Carlsbad, CA, USA) was used as a probe for this analysis and manufacturer protocols were used for hybridization and detection steps. Two mice (n = 68 metaphase cells per genotype) from each genotype were analyzed.

Immunoblotting and reverse-phase protein array (RPPA)

Immunoblotting was performed with standard procedures. Antibodies are listed in Supplementary Table 2. For RPPA, protein lysates were prepared according to protocols provided by the MD Anderson Cancer Center Functional Proteomics RPPA Core (supported by MD Anderson Cancer Center Support Grant #5 P30 CA016672-40). The RPPA core performed the array and analyzed the data. Log₂-transformed data, median-centered, were used to generate the heatmap.

In vivo tumorigenesis

Murine tumors dissected into ~27-mm³ cubes and placed into serum-free Media 199 (#11150-067, Gibco) supplemented with 1% antibiotic/antimycotic (#A5955, Sigma) prior to implantation into SCID/Beige (#250, Charles River Laboratories) or CB17 (#CB17SC, Taconic Biosciences) mice. Allografts implanted via flank incision and closed with VetBond (#14695B, 3 M).

Dissociated PDX samples were transduced with lentivirus for 2.5 h. One million PDX cells/mouse were resuspended in 100 μL matrigel (#354234, Corning) and implanted into the left hindlimb of adult SCID/Beige mice. Volumes calculated as π/6 x [(L + W)/2]3 ⁶³; tumor volume reported as the difference between the injected and contralateral, uninjected limb.

RNA isolation and gene expression analysis

RNA was isolated using a Qiagen miRNeasy Mini (#217004, Valencia, CA, USA) or Micro (#217084) (for sorted tumor populations and primary tumorsphere experiments) kit according to the manufacturer instructions. Reverse transcription was performed with Superscript III First Strand cDNA Synthesis Kit using random hexamer primers (ThermoFisher, #180800551). SYBR primers and Taqman probes used are listed in Supplementary Table 1. Data normalized to Actb/ACTB or 18S rRNA expression.

For microarray analysis changes between ASP^cKO and ASP^WT tumors, amplified cDNA was prepared using an Ovation Pico WTA System V2 (#3302, Nugen, San Carlos, CA) and analysis was performed on a Mouse 2.0 ST microarray (#902118, Affymetrix, Santa Clara, CA, USA)²⁰. Data were imported into Partek Genome Suite 6.6, visualized by principal component analysis, batch corrected, and statistically tested using an unequal variance t test. Unannotated transcripts were filtered out and imported into STATA/MP 14.2 and P values adjusted for multiple comparisons by the Benjamini–Hochberg FDR method⁶⁶. Volcano plots were also produced in STATA/MP 14.2 by plotting the log₁₀ transformed P value from the unequal variance t test against the log ratio of expression.

CIBERSORT analysis for predicted differences in immune cell compartments within ASP^WT and ASP^cKO tumors was performed with the LM22 leukocyte gene signature⁶⁷.

Gene expression changes between shDBX1-transduced and shScrambled-transduced SJRHB015271_X1 cells, Clariom S human microarrays were used. In total, 125 ng RNA was processed using ThermoFisher (Affymetrix) Whole Transcript (WT) Plus assay kit (#902280). Labeled cDNA included in a hybridization mix incubated on the Clariom S human array for 16 h at 45 °C while rotating at 60 rpm. Cartridges were stained and washed on the Gene Chip FS450 fluidics station and scanned on the Gene Chip Scanner 3000 7G. Data were imported, visualized by principal component analysis, statistically analyzed, and rendered as described above.

Low-input RNA sequencing was performed for gene expression changes between flow cytometry-sorted tumor populations from the ASP^cKOP7^cKO and ASP^cKO tumors. In all, 5 ng RNA was used in the Tecan Ovation RNA sequencing system v2 protocol as written to create SPIA (single primer isothermal amplification)-cDNA. Once purified, 500 ng of the sample was sheared (Covaris LE220 focused ultra-sonicator (96 microtube-50 AFA fiber plate, target base pair size of 300; shearing settings: Peak Incident Power (W) of 450, duty factor of 15%, cycles per burst 1000 with a 100 s treatment time)). Libraries were created with sheared cDNA and the KAPA Hyper-Prep kit (#KK8604, Roche). Four PCR cycles used for the cDNA amplification step and the protocol was followed according to the manufacturer’s instructions. UDI DNA indexes from Illumina (#20022370) were used.

Total stranded RNA sequencing data were processed by the internal AutoMapper pipeline. Raw reads were first trimmed (Trim-Galore version 0.60), mapped to mouse genome assembly (GRCm38) (STAR v2.7)⁶⁸ and then the gene level values were quantified (RSEM v1.31) based on GENCODE annotation (vM22). Low count genes removed from analysis using a CPM cutoff corresponding to a count of ten reads and only confidently annotated (level 1 and 2 gene annotation) and protein-coding genes are used for differential expression analysis. Normalization factors were generated using the TMM method; counts then transformed using voom. Transformed counts analyzed using the lmFit and eBayes functions (R limma package version 3.42.2). The significantly up- and downregulated genes were defined by at least fold change >4 and P value <0.05.

For gene ontology (GO) analyses, Gene Set Enrichment Analysis (GSEA) or functional annotation clustering using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, version 6.8) was used. For GSEA, the GSEA software⁶⁹ version 4.0 was used and based on the pre-ranked option. Normalized enrichment score used for visualization. For DAVID⁷⁰, functional annotation clustering using the GOTERM_BP_FAT selection was performed. Top ten unique GO terms visualized and represented as the −log₁₀ P value.

Statistics

Sample size and replicates are listed in Figure legends. Error bars reported as mean ± SEM and statistical significance determined by using two-tailed, unpaired Student’s t test for pairwise comparisons unless otherwise indicated. Kaplan–Meier survival analyses were performed by log-rank (Mantel–Cox) tests. P values below 0.05 considered significant and indicated by the following: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. GraphPad Prism 9 was used for statistical tests. Statistical analyses of gene expression data described above.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Microarray and RNAseq data generated are deposited in the Gene Expression Omnibus (GSE166906). St. Jude ProteinPaint RNA sequencing data accessed through their open-resource page (https://pecan.stjude.cloud)²⁹. RNA sequencing data from the US National Cancer Institute RMS dataset accessed from³⁰. RNA sequencing and dependency data (CERES score)²⁸ from the Broad Institute’s Cancer Dependency Map accessed from the 20Q3 data deposit (https://depmap.org/portal/). St. Jude and US NCI RMS core-regulatory circuit data are previously published and referenced, respectively^34,35. Inter-species sequence conservation for the DBX1 promoter was performed using the Evolutionary Conserved Regions database (https://ecrbrowser.dcode.org)⁷¹. Transcription factor binding sites found within the murine Dbx1 promoter were mined from the JASPAR 2016 database (https://jaspar2016.genereg.net)⁷². All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files, Source Data file, or from the corresponding author, Mark E. Hatley (mark.hatley@stjude.org), upon reasonable request. A Reporting Summary for this article is available in a Supplementary Information file. Source data are provided with this paper.

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Acknowledgements

We thank the SJCRH Shared Resources including Flow Cytometry and Cell Sorting (Richard Ashmun), Veterinary Pathology Core (Peter Vogel, Pam Johnson), Cell and Tissue Imaging Electron Microscopy Services (Cam Robinson), the Core Cytogenetic Laboratory (Marc Valentine, Virginia Valentine), the Hartwell Center and Functional Genomics (Geoff Neale, Melanie Loyd), and the Center for Applied Bioinformatics (Gang Wu). We thank Johnathan Dallman, Jonathan Go, D. Wood Kimbrough, and Lauren Goudie for technical assistance. Research reported in this publication supported by the National Cancer Institute of the National Institutes of Health under Award Numbers R01CA216344 and R01CA251436 (MEH) and F31CA250398 (MB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The Hatley laboratory is also supported by grants from The V Foundation for Cancer Research, the Rally Foundation for Childhood Cancer Research and Open Hands Overflowing Hearts award number 20IC23 (MEH), St. Jude Cancer Center Support Grant (P30 CA21765), and American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital. K.E.G. is supported by a Damon Runyon-Sohn Pediatric Cancer Fellowship (DRSG-30-19).

Author information

Jason A. Hanna
Present address: Purdue Center for Cancer Research, Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
Catherine J. Drummond
Present address: Department of Pathology, University of Otago, Dunedin, Otago, New Zealand

Authors and Affiliations

Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA
Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Myron K. Evans, Kristin B. Reed, Madeline Bush, Jason A. Hanna, Catherine J. Drummond, Matthew C. Maguire, Patrick J. Leavey & Mark E. Hatley
Rhodes College, Memphis, TN, 38112, USA
Kristin B. Reed
St. Jude Graduate School of Biomedical Sciences, Memphis, TN, 38105, USA
Madeline Bush
Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA
David Finkelstein
Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA
Hongjian Jin & Patrick A. Schreiner
Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA
Jerold E. Rehg

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Casey G. Langdon
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Katherine E. Gadek
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Matthew R. Garcia
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Madeline Bush
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Jason A. Hanna
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Contributions

Conceptualization—C.G.L. and M.E.H.; methodology—C.G.L., M.R.G., K.E.G., M.K.E., J.A.H., and M.E.H.; formal analysis—C.G.L., J.E.R., and M.E.H.; investigation—C.G.L., K.E.G., M.R.G., M.K.E., K.B.R., M.B., J.A.H., C.J.D., P.A.S., P.J.L., and M.C.M.; data curation—D.F. and H.J.; writing: original draft—C.G.L. and M.E.H.; writing: reviewing and editing—all authors; visualization—C.G.L. and M.E.H.; supervision—M.E.H.; funding acquisition—M.E.H.

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Correspondence to Mark E. Hatley.

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Langdon, C.G., Gadek, K.E., Garcia, M.R. et al. Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity. Nat Commun 12, 5520 (2021). https://doi.org/10.1038/s41467-021-25829-4

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Received: 05 April 2021
Accepted: 01 September 2021
Published: 17 September 2021
DOI: https://doi.org/10.1038/s41467-021-25829-4

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