MAPK4 promotes triple negative breast cancer growth and reduces tumor sensitivity to PI3K blockade

About 15–20% of breast cancer (BCa) is triple-negative BCa (TNBC), a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN signaling pathway are common in TNBC. However, the therapeutic impact of PI3K inhibitors in TNBC has been limited and the mechanism(s) underlying this lack of efficacy remain elusive. Here, we demonstrate that a large subset of TNBC expresses significant levels of MAPK4, and this expression is critical for driving AKT activation independent of PI3K and promoting TNBC cell and xenograft growth. The ability of MAPK4 to bypass PI3K for AKT activation potentially provides a direct mechanism regulating tumor sensitivity to PI3K inhibition. Accordingly, repressing MAPK4 greatly sensitizes TNBC cells and xenografts to PI3K blockade. Altogether, we conclude that high MAPK4 expression defines a large subset or subtype of TNBC responsive to MAPK4 blockage. Targeting MAPK4 in this subset/subtype of TNBC both represses growth and sensitizes tumors to PI3K blockade. PI3K inhibitors have limited efficacy in triple negative breast cancer (TNBC). Here, the authors show that MAPK4 activates AKT independent of PI3K and thus promotes tumour growth in a subset of TNBC and that MAPK4 inhibition sensitizes to PI3K blockade in these tumours.’

T he AKT/mTOR signaling pathway plays critical roles in regulating cell proliferation, survival, and metabolism. In the canonical pathway, PI3 kinase (PI3K) catalyzes phosphatidylinositol-3,4,5-triphosphate (PIP3) production, and PIP3 binds and recruits AKT to the plasma membrane for activation [1][2][3] . Phosphorylation of AKT at both Thr308 (T308) and Ser473 (S473) is requisite for full AKT activation. mTOR forms two different complexes, mTORC1 and mTORC2. AKT activates mTORC1, which integrates extracellular stimuli and nutrient signals to modulate cell growth, autophagy, and metabolism 4 . In contrast, mTORC2 is the main S473 kinase of AKT 5 , while PDK1 is the major T308 kinase 6 . The PI3K/AKT/ mTOR signaling pathway is crucial in regulating tissue homeostasis, and its dysregulation can cause various pathological conditions including cancers.
About 15-20% of all breast cancers (BCa) are triple-negative (TNBC) [7][8][9] , a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN/AKT signaling pathway are common in TNBC 10 . The FDA has recently approved the α-isoform-specific PI3K inhibitor Piqray (Alpelisib) to be used in combination with fulvestrant to treat hormone receptor-positive, HER2-negative, PIK3CA-mutated, advanced or metastatic BCa following progression on or after an endocrine-based regimen. However, PI3K inhibitors have shown only limited therapeutic responses when use to treat TNBC 11,12 .
MAPK4 is an atypical MAPK that has not been well studied [13][14][15] . We recently reported that MAPK4 is a key oncogenic kinase promoting cancer via non-canonical activation of AKT/mTOR independent of PI3K/PDK1 16 . Here we report that MAPK4 is highly expressed in a large subset of TNBC. MAPK4 overexpression is sufficient to drive oncogenic outcomes, while repressing MAPK4 in the MAPK4-high human TNBC cells greatly inhibits AKT activation, cell proliferation, and anchorageindependent growth in vitro, and xenograft growth in vivo. The ability of MAPK4 to directly activate AKT suggests that elevated MAPK4 expression should reduce tumor sensitivity to PI3K blockade. In accord with this, knockdown/knockout of MAPK4 in the MAPK4-high TNBC cells and xenografts sensitized them to PI3K inhibition. These results identify MAPK4 as a promising therapeutic target for TNBC and its potential in combined therapy with PI3K inhibition.

Results
MAPK4 is highly expressed in a significant subset of human TNBC. Analysis of 817 gene expression profiles in The Cancer Genome Atlas (TCGA) 17 revealed that MAPK4 expression is elevated in 30% or more of basal-like BCa (Fig. 1a), 70-80% of which are TNBC [18][19][20][21][22][23] . In contrast, much lower MAPK4 expression was detected in the other luminal A, luminal B, HER2amplified, and normal-like BCa types. We also analyzed MAPK4 expression in the Baylor College of Medicine BCa patient-derived xenograft (PDX) collection with completed RNA-Seq data (n = 92, https://pdxportal.research.bcm.edu/, public and private data combined), the majority of which are TNBC (n = 69, Fig. 1b,  c). We also observed that MAPK4 is highly expressed in a large subset of these TNBC PDX models. Altogether, these data support that MAPK4 is expressed at significant levels in a large subset of TNBC/basal-like BCa.
MAPK4 activates AKT in human TNBC cells. To assess MAPK4 biology in human TNBC, we first surveyed its expression in multiple commonly used TNBC lines, including HS578T, MDA-MB-231, HCC1937, SUM159, MDA-MB-468, HCC1395, and HCC1806 cells, as well as in the "normal" human mammary epithelial MCF10A cells. We observed high levels of endogenous MAPK4 (MAPK4-high) expression in the MDA-MB-231, HS578T, and HCC1937 cells, followed by a lower MAPK4 expression in SUM159 cells (Fig. 2a). In contrast, MDA-MB-468, HCC1806, HCC1395, and the "normal" MCF10A cells express low-to-nondetectable levels of MAPK4. As an initial test of the impact of MAPK4 expression, we performed knockdown of MAPK4 (lentiviral shRNA) in the MAPK4-high MDA-MB-231, HS578T, and HCC1937 cells as well as in SUM159 cells. We observed that MAPK4 knockdown in all four TNBC cell lines repressed AKT phosphorylation and inhibited its activation, as evidenced by inhibition of GSK3β phosphorylation (Fig. 2b). We also overexpressed MAPK4 in SUM159, MDA-MB-468, HCC1395, HCC1806, and MCF10A cells in a Dox-inducible manner. In accord with loss of function results, MAPK4 overexpression induced AKT phosphorylation and activation in these cells ( Fig. 2c and see below, Fig. 4d). In agreement with our previous observation in other types of human cancers 16 , these results indicate that MAPK4 plays essential roles in promoting AKT phosphorylation/activation in the MAPK4-high TNBC cells.
To further confirm and extend these observations, we used CRISPR/Cas9 technology to generate MAPK4 null BCa cell lines as previously described 16 . As expected, genetic ablation of MAPK4 in both MDA-MB-231 and SUM159 cells markedly inhibited AKT phosphorylation and activation (Fig. 2d). Finally, we demonstrated that MAPK4 overexpression rescued AKT phosphorylation in MAPK4-knockout MDA-MB-231 and SUM159 cells (Fig. 2e). Altogether, these data support a critical role of MAPK4 in promoting AKT phosphorylation/activation in human TNBC cells.
MAPK4 promotes TNBC cell growth in vitro. We next determined the impact of MAPK4 on TNBC cell growth. MAPK4 knockdown in the MAPK4-high human TNBC HCC1937 and HS578T cells, as well as in SUM159 cells, greatly inhibited their growth, including anchorage-independent growth in vitro (Fig. 3a, c-e). Accordingly, overexpression of MAPK4 promoted SUM159, HCC1395, and HCC1806 cell growth, as well as the anchorage-independent growth of SUM159 cells (Fig. 3b, g). Increased BrdU incorporation confirmed the enhanced proliferation of SUM159 cells in response to Dox-induced MAPK4 overexpression (Fig. 3h). These data support a crucial role of MAPK4 in promoting TNBC cell growth.
Human TNBC MDA-MB-231 cells have properties of cancer progenitor cell populations 24 . Accordingly, while MAPK4 knockdown in MDA-MB-231 cells did not significantly affect their proliferation ( Supplementary Fig. 1a), it greatly repressed their anchorage-independent growth (Fig. 3f) and suppressed mammosphere formation (Fig. 3i). These results further support MAPK4 tumor-promoting activity in TNBC and shed light on a potential role of MAPK4 in regulating TNBC progenitor cell biology.
We have previously shown that MAPK4 overexpression transformed the "normal" prostate epithelial PNT1A cells into anchorage-independent growth in vitro 16 . To further assess the oncogenic activity of MAPK4 in mammary epithelial cells, we investigated MAPK4 activity in transforming "normal" human mammary epithelial MCF10A cells. As expected, MAPK4 overexpression similarly activated AKT and transformed MCF10A cells into anchorage-independent growth, further supporting the oncogenic activity of MAPK4 in mammary epithelial cells (Fig. 4d, e).
MAPK4 promotes insulin and EGF-induced AKT activation in a parallel action of PI3K. Due to the lack of conserved T-x-Y motif (S-E-G for MAPK4), there are no identified MAPK kinases (MAPKKs), the dual Ser/Thr and Tyr kinases, to phosphorylate and activate MAPK4. Type I P21 activated kinases 1, 2, and 3 (PAK1/2/3) have been shown to phosphorylate S186 (S189 on the closely related MAPK6) and activate the MAPK4/MAPK6-MK5 signaling cascade 25 . However, a previous study revealed no correlation between cellular MAPK4/MAPK6 phosphorylation and the extracellular stimuli or stress conditions examined 26 , and the impact of this pathway on MAPK4-AKT signaling remains unknown. To determine whether extracellular stimuli can activate the MAPK4-AKT signaling cascade, we first examined AKT phosphorylation in insulin (100 nM) stimulated serum-starved MDA-MB-231 and HS578T cells with Dox-induced knockdown of MAPK4 (ishMAPK4) or control (iNT). Insulin treatment of the control TNBC cell lines greatly stimulated AKT phosphorylation, which lasted at least 2 h. In contrast, knockdown of MAPK4 significantly reduced insulin-stimulated AKT phosphorylation at 10 min after treatment, and rapidly decreased such phosphorylation to basal levels within 1-2 h (Fig. 5a).
To investigate how PI3K pathway inhibition affects MAPK4-AKT signaling, we examined AKT phosphorylation/activation status in insulin (100 nM) stimulated serum-starved SUM159 cells with Dox-induced knockdown of MAPK4 or control. These cells were also pre-treated with PI3K inhibitors Pictilisib (20 nM), Alpelisib (100 nM), or DMSO vehicle control. In accord with the prediction of parallel pathways for AKT activation, either MAPK4 knockdown or PI3K inhibition (Pictilisib or Alpelisib) alone exhibited partial effects, while concurrent MAPK4 knockdown and PI3K inhibitor treatment robustly blocked insulininduced AKT phosphorylation/activation (Fig. 5b).
To further expand these observations, we performed a similar study in EGF (200 ng/ml) stimulated serum-starved MDA-MB-231 and SUM159 cells with Dox-induced knockdown of MAPK4 or control. These cells were also pre-treated with PI3K inhibitors Pictilisib (20 nM), Alpelisib (100 nM), or DMSO vehicle control. Similar to insulin treatment, EGF treatment of the control TNBC cell lines greatly promoted AKT phosphorylation and activation. Again, while either knockdown of MAPK4 or PI3K inhibitor (Pictilisib or Alpelisib) treatment alone exhibited partial effect, concurrent knockdown of MAPK4 and PI3K inhibitor treatment robustly blocked EGF-induced AKT phosphorylation/activation (Fig. 5c). Altogether, these data strongly support that extracellular stimuli including insulin and EGF can activate the MAPK4-AKT signaling axis in a pathway that parallels the action of PI3K.
AKT activation is critical for MAPK4 tumor-promoting activity in TNBC. We previously demonstrated that AKT activation is critical for the tumor-promoting activity of MAPK4 16,27 . To further confirm this in TNBC, we examined how the AKT inhibitors MK2206, GSK2141795, and/or GDC-0068 affect growth of the engineered HCC1395, HCC1806, and SUM159 cells with 0.5 µg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl). These AKT inhibitor treatments largely abolished the MAPK4 activities in enhancing the growth of HCC1395, HCC1806, and SUM159 cells as well as MAPK4 activities in promoting the anchorage-independent growth of SUM159 cells (Fig. 6a-d). These data confirm that, as we showed in other cancer types 16,27 , AKT is also a key node for mediating MAPK4 activity in promoting TNBC.
Knockdown of MAPK4 both significantly repressed MDA-MB-231, SUM159, HS578T, and HCC1937 cell growth and sensitized them to both Pictilisib and Alpelisib treatments ( Fig. 7a-d; Supplementary Fig. 1b). We further confirmed that knockdown of MAPK4 also sensitized SUM159 cells to another commonly used PI3K inhibitor LY294002 (2 μM), and the effects were comparable to treatments using Pictilisib (1 μM) and Alpelisib (0.5 μM, Fig. 7e). Interestingly, after 10 days of culture in the clonogenic assay settings (cells plated at low density for clonal growth of individual cells), the MAPK4-knockdown SUM159 and iNT  MDA-MB-231 cells partially regained AKT phosphorylation/ activation, presumably due to PI3K-AKT pathway activation for individual cell growth/survival. In accord with this, AKT phosphorylation/activation in these MAPK4-knockdown cells was highly sensitive to PI3K blockade (0.5 μM Alpelisib), further confirming MAPK4-AKT as the essential pathway for regulating TNBC sensitivity to PI3K inhibition (Fig. 7f).
To define whether MAPK4 overexpression makes TNBC cells less sensitive to PI3K blockade, we also performed colony formation assays on the engineered SUM159, MDA-MB-468, HCC1395, and HCC1806 cells with 0.5 µg/ml Dox-induced ectopic expression of MAPK4 (iMAPK4) or control (iCtrl). We similarly treated these cells with increasing doses of PI3K inhibitors Pictilisib, Alpelisib, or DMSO vehicle control. As expected, MAPK4 overexpression both significantly promoted SUM159, MDA-MB-468, HCC1395, and HCC1806 cell growth and reduced their sensitivity to both Pictilisib and Alpelisib (Fig. 8a-d; Supplementary Fig. 1c). Accordingly, the AKT phosphorylation/activation was markedly maintained in the MAPK4-overexpressing SUM159 and HCC1806 cells under PI3K inhibitor treatments (compared to similarly treated iCtrl cells), further confirming MAPK4-AKT as the essential pathway for regulating TNBC sensitivity to PI3K inhibition (Fig. 8e).
Altogether, these results support a crucial role of MAPK4 in not only driving TNBC cell growth but also reducing their sensitivity to PI3K inhibition.
MAPK4-knockout TNBC xenografts are sensitive to PI3K inhibitor Alpelisib. To further assess whether MAPK4 blockage can sensitize TNBC tumors to PI3K inhibition, we examined how MAPK4 knockout affects MDA-MB-231 cell and xenograft growth and sensitivity to PI3K blockade. Consistent with the MAPK4-knockdown data (Fig. 3f and Fig. 9b, d) MDA-MB-231 cells formed fewer colonies and were more sensitive to PI3K inhibitors LY294002, Pictilisib, and Alpelisib in the soft-agar assays (Fig. 10a). Ectopic MAPK4 expression both promoted the anchorage-independent growth of these MAPK4-KO cells and largely rescued their growth in the presence of PI3K inhibitors (Fig. 10a).
We next performed xenograft studies in SCID mice using parental and MAPK4-KO MDA-MB-231 cells. Consistent with our above data from the MAPK4-knockdown MDA-MB-231 xenografts (Fig. 4a), knockout of MAPK4 also significantly repressed MDA-MB-231 xenograft growth (Fig. 10b, c). The PI3K inhibitor Alpelisib showed minimal effect on the growth of the parental MDA-MB-231 xenografts. In contrast, Alpelisib significantly inhibited the growth of MAPK4-KO MDA-MB-231 xenografts (Fig. 10b, c), supporting that loss of MAPK4 sensitizes TNBC tumors to PI3K inhibition in vivo.

Discussion
Profiling human tumors show that MAPK4 is highly expressed in a large subset of TNBC. We estimate that this includes 30% or more of basal-like BCa/TNBC, with lesser amounts in other Twenty hours later, the cells were serum-starved overnight followed by treatments with 100 nM insulin for the indicated time (minutes). The cell lysates were then prepared and used in western blots. b Western blots on SUM159-ishMAPK4 and -iNT cells that were similarly treated as described above. These cells were also pre-treated with Pictilisib or DMSO control for 2 h before 100 nM insulin stimulation. c Western blots on MDA-MB-231 and SUM159 cells with Dox-inducible knockdown of MAPK4 (ishMAPK4) or control (iNT) that were similarly treated as described above. These cells were pre-treated with PI3K inhibitors Pictilisib, Alpelisib, or control for 2 h followed by 200 ng/ml EGF stimulation for the indicated time. Data are representative of at least three independent experiments. subtypes. In accord with our earlier results in other cancers 16,27 , MAPK4 knockdown or knockout in these MAPK4-high TNBC cell lines greatly repressed AKT activation, cell growth, and xenograft growth. In contrast, MAPK4 promoted cell migration in the wound healing assay in only one out of four TNBC cell lines tested (Supplementary Fig. 2). Therefore, unlike the growthpromoting activity, MAPK4's ability to promote cell motility appears cell-context dependent.
Our previous results also showed that MAPK4 expression can transform the normal prostate epithelial PNT1A cells into both anchorage-independent growth in vitro 16 and orthotopic xenograft tumor growth in vivo (unpublished observation). Our current results show that MAPK4 can transform normal human mammary epithelial MCF10A cells into anchorage-independent growth in vitro but failed to transform them into tumor growth in vivo six months after initial inoculation into mammary fat pad ( Fig. 4e and unpublished observation). Overall, these data define MAPK4 as a bona fide oncogene and indicate that the MAPK4high subset may represent a unique subtype of TNBC that would be sensitive to MAPK4 inhibition. Interestingly, five out of the  seven TNBC cell lines that we have tested are either PTEN-null (HCC1395, HCC1937, and MDA-MB-468 cells with frameshift mutation or homodeletion of PTEN) or PIK3CA (SUM159) or PIK3R1 mutated (HS578T). We are investigating the detailed molecular mechanism underlying the maintenance of the MAPK4-addiction phenotype in these TNBC cells. Although there are currently no identified specific MAPK4 inhibitors, the current results provide further impetus for their development and for future studies to critically examine their efficacy, either as monotherapy or in combination with chemotherapy or radiation therapy, in treating MAPK4-high TNBC.
MAP kinases (MAPKs) play critical roles in mediating cell response to extracellular signals. In the canonical MAPK pathway, MAPKs are phosphorylated at both S/T and Y residues in the conserved T-x-Y motif and activated by MAPK kinases (MAPKKs), the dual Ser/Thr and Tyr kinases. In MAPK4, this T-x-Y motif is replaced by S-E-G (aa186-188), which lacks the key Y residue. Therefore, there is no identified MAPKK to phosphorylate/activate MAPK4. Furthermore, MAPK4 phosphorylation was not associated with extracellular stimuli or stress conditions examined 26 , leading to a vague conception that MAPK4 activation is not subjected to such conditions. In contrast, our study demonstrated that the MAPK4-AKT signaling cascade can be activated by both insulin and EGF, two key factors regulating physiology and diseases, including cancers. Further studies are needed to assess the detailed mechanism underlying how extracellular signals activate the MAPK4-AKT pathway. Despite the success of the PI3K inhibitor Alpelisib in treating hormone receptor-positive, HER2-negative, PIK3CA-mutated, advanced or metastatic BCa, the therapeutic effect of PI3K inhibitors in TNBC is limited 11,12 . Both intrinsic and acquired resistance may be responsible for the lack of efficacy of PI3K inhibition in TNBC. Our identified PI3K-independent MAPK4-AKT signaling axis may provide a pathway for MAPK4-high TNBC tumor intrinsic resistance to PI3K inhibitors. Indeed, without exception, knockdown or knockout of MAPK4 in the MAPK4-high MDA-MB-231, HCC1937, HS578T, as well as MAPK4-medium SUM159 cells sensitized them to PI3K inhibition in vitro. Knockout of MAPK4 in the MDA-MB-231 xenografts both repressed their growth and sensitized them to Alpelisib in vivo. These data support the prediction that inhibiting MAPK4 should sensitize MAPK4-high TNBC to PI3K inhibition. Future studies are needed to test this prediction using MAPK4-specific inhibitors in combination with PI3K inhibitors in both pre-clinical and clinical settings. It will also be interesting to assess whether MAPK4 expression/activity is induced in TNBC that gain resistance to PI3K inhibition and whether targeting MAPK4 will overcome this acquired resistance.
We previously demonstrated that AKT activation is essential for mediating the tumor growth-promoting effects of MAPK4 16 . Our current data further confirmed the indispensable role of AKT activation in mediating MAPK4 activity in promoting TNBC growth. This leads to the question of whether MAPK4-high expression status, independent of PIK3CA/PTEN/AKT1 alteration, will define TNBC tumors with a better response to AKT blockade. This is particularly important since a recent phase 3 IPATunity130 trial testing the AKT inhibitor ipatasertib (GDC-0068) plus paclitaxel in patients with PIK3CA/AKT1/PTENaltered TNBC did not significantly improve progression-free survival when compared with placebo plus paclitaxel treatment group 28 . We are actively pursuing this research direction.  Viruses were harvested and applied for cell infection as described before 16 . The established cell lines were then expanded and stocked for further assays. The pInducer10 based constructs were used for lentivirus-mediated Dox-inducible knockdown of MAPK4 (iG2 and iG4) or control (iNT). The cells were induced with up to 4 μg/ml Dox for at least 3 days to obtain significant knockdown of MAPK4. The pInducer20-YF based constructs were used for lentivirus-mediated Dox-inducible overexpression of MAPK4 (iMAPK4) or control (iCtrl). The cells were treated with up to 1 μg/ml Dox for at least 2-3 days for ectopic overexpression of MAPK4. The MAPK4-knockout (KO) SUM159 and MDA-MB-231 cell lines were created using a similar protocol as we described previously 16 . Knockout of MAPK4 in each single clone was verified by genomic sequencing and western blots. The pCDH based lentiviral constructs were used for lentivirus-mediated stable overexpression of MAPK4 in the MAPK4-KO cell as described before 16 .
Western blot. Cell lysates were prepared in RIPA buffer and protein concentrations were quantified using a Pierce BCA protein assay kit. An equal amount of protein (5-20 μg) was used in western blot analysis.
Cell proliferation assays. We used three approaches to assess cell proliferation, including direct cell counting, crystal violet staining-based cell proliferation assay, and BrdU incorporation assay as previously described 16 . When applicable, the kinase inhibitor(s) or vehicle control was added during the initial setup and replenished in fresh media every 3 days.
Colony formation assay. 1000-2500 single cells were suspended and seeded into each well of 6-or 12-well plates. The cells were then treated with the indicated inhibitors or vehicle control in triplicates for 10-21 days. Cells were then fixed with 10% (w/v) formaldehyde for 15 min and stained with 0.05% (w/v) crystal violet supplemented with 10% ethanol and 10% methanol for 20 min at room temperature. After a final wash of three times with distilled water, the plates were airdried and scanned using a Canon scanner. The cell colonies were quantified either using ImageJ (area%) or by directly measuring absorbance (570 nm) of the solved crystal violet in 10% acetic acid. In the latter case, a background reading of 0.10 (the average OD reading of stained empty wells from multiple independent experiments using the same protocol) was reduced from all data points to remove background noises.
Soft-agar colony formation assay. Soft-agar colony formation assays were performed as described before 16 . 4 μg/ml or up to 1 μg/ml Dox were used for inducing knockdown or overexpression of MAPK4 in the indicated engineered cells. When applicable, PI3K inhibitors LY294002, Alpelisib, or Pictilisib at the indicated concentrations were added during the initial setup and replenished in fresh media every week. The colony numbers were counted and quantified after 3-4 weeks.
Scratch wound healing assay. Scratch wound healing assays were performed to evaluate cell migration on 6-well plates (100,000 cells/well). Cell monolayers at confluency were scratched with a 200 μl tip. Wound closure was analyzed at 8 and 24 h after scratch.
Xenograft tumor models. Female SCID/beige mice at 8-10 weeks old from Envigo were used in the xenograft studies. Mice were housed in a pathogen-free facility at Baylor College of Medicine. MDA-MB-231 and HCC1937 cells (2 × 10 6 ) with Dox-inducible knockdown of MAPK4 (ishMAPK4) vs. control (iNT) were injected into mammary fat pads (iNT, left side; ishMAPK4, right side). SUM159 cells with Dox-inducible expression of MAPK4 (iMAPK4) or control (iCtrl) were similarly injected. Mice began receiving 4 mg/ml (for inducible knockdown) or 0.5 mg/ml (for inducible overexpression) Dox in 1-10% sucrose in drinking water on the day of tumor injection and throughout the studies. Tumors were monitored/measured every week, and tumor volumes were calculated as Vol = 0.52 × abc (a, b, c: the maximum length of each dimension of the tumor). The wild type and MAPK4-KO MDA-MB-231 cells (1 × 10 6 ) were similarly used in the Alpelisib treatment studies. When tumors reach significant sizes (average of 100-200 mm 3 ), Alpelisib (20 mg/kg) was delivered to mice daily through oral gavage. Tumors were similarly monitored, and tumor volume calculated every week. All tumors were harvested as indicated and weighed. Average tumor weight was compared among different groups for statistical relevance using the unpaired two-tailed Student's t test. P < 0.05 is considered statistically significant.
Study approval. All animal studies were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.
Statistics. TCGA RNA-seq data were obtained from the Broad Institute's Firehose data portal (https://gdac.broadinstitute.org). P value was calculated by two-sided t test on log2-transformed expression values. The statistical relevance in the cellculture studies and xenograft tumor studies was analyzed using the unpaired twotailed Student's t test. When multiple comparisons were made, one-way or two-way ANOVA followed by Dunnett's multiple comparisons test or Sidak's multiple comparisons test was performed using GraphPad Prism, 9.3. P < 0.05 was considered significant.