PTPN11 encodes the Shp2 non-receptor protein-tyrosine phosphatase implicated in several signaling pathways. Activating mutations in Shp2 are commonly associated with juvenile myelomonocytic leukemia but are not as well defined in other neoplasms. Here we report that Shp2 mutations occur in human acute myeloid leukemia (AML) at a rate of 6.6% (6/91) in the ECOG E1900 data set. We examined the role of mutated Shp2 in leukemias harboring MLL translocations, which co-occur in human AML. The hyperactive Shp2E76K mutant, commonly observed in leukemia patients, significantly accelerated MLL-AF9-mediated leukemogenesis in vivo. Shp2E76K increased leukemic stem cell frequency and affords MLL-AF9 leukemic cells IL3 cytokine hypersensitivity. As Shp2 is reported to regulate anti-apoptotic genes, we investigated Bcl2, Bcl-xL and Mcl1 expression in MLL-AF9 leukemic cells with and without Shp2E76K. Although the Bcl2 family of genes was upregulated in Shp2E76K cells, Mcl1 showed the highest upregulation in MLL-AF9 cells in response to Shp2E76K. Indeed, expression of Mcl1 in MLL-AF9 cells phenocopies expression of Shp2E76K, suggesting Shp2 mutations cooperate through activation of anti-apoptotic genes. Finally, we show Shp2E76K mutations reduce sensitivity of AML cells to small-molecule-mediated Mcl1 inhibition, suggesting reduced efficacy of drugs targeting MCL1 in patients with hyperactive Shp2.
MLL rearrangements are present in ~20% of pediatric acute myeloid leukemia (AML) and can be as high as 80% of infant patients with acute lymphocytic leukemia,1 and are generally associated with a poor outcome.2 Rearrangements of the MLL locus generate potent oncogenic fusion proteins that retain the N-terminus of the MLL protein but replace the C-terminus with one of >60 different partner proteins that can recruit transcriptional activation complexes.3, 4, 5, 6 The resultant deregulated transcriptional activation mediated by MLL fusion proteins blocks hematopoietic differentiation through the sustained expression of the posterior HOXA gene cluster, namely HOXA9.7 Interestingly, MLL leukemias display a relatively stable genome compared with other leukemic subtypes but still carry other genetic lesions at low frequency.8 Type-I mutations involving the Ras pathway are present in ~37% of MLL rearranged leukemias including mutations within NRAS, KRAS, NF1 and PTPN11,9 consistent with the idea that pathological AML requires both type-I and type-II mutations.10 Indeed, oncogenic NRASG12V or FLT3-ITD can significantly accelerate MLL fusion protein-mediated leukemogenesis in vivo.11, 12, 13 Although these mutations strongly cooperate with MLL fusion proteins to promote leukemogenesis, little is understood about the molecular mechanisms used by type-I mutations.
PTPN11 encodes the ubiquitously expressed SHP2 non-receptor protein-tyrosine phosphatase involved in the RAS, JAK-STAT, PI3K and other pathways.14, 15 Mutations in PTPN11 are found in ~50% of patients with Noonan syndrome and in ~37% of patients with hematologic malignancies such as juvenile myelomonocytic leukemia, acute lymphocytic leukemia and AML.16, 17, 18, 19 Recent genome-wide sequencing analyses have identified PTPN11 mutations in AML patients, indicating this may function in a cooperative manner.20, 21 Shp2 positively regulates signal transduction pathways downstream of receptor tyrosine kinases, such as Kit, where it is essential for hematopoietic stem and progenitor cells.22, 23 Hematopoietic progenitors require Shp2 for STAT5 activation and upregulation of Mcl1 and Bcl-xL.24, 25 In leukemia, expression is often elevated and Shp2 can associate with FLT3-ITD, leading to activation of STAT5. Shp2 co-localizes with STAT5 to activate expression of Bcl-xL protecting against cell death.26, 27 PTPN11 mutations result in amino acid changes resulting in disrupted autoinhibition and hyperactive Shp2 enzymatic activity.17, 28, 29, 30 Gain-of-function mutations in Shp2 result in cytokine hypersensitivity in hematopoietic progenitor cells.31 In mice, gain-of-function Shp2 mutations leads to a juvenile myelomonocytic leukemia-like fatal myeloproliferative disease, whereas an inducible mutant Shp2 knockin mouse model progresses to AML, as well as B- and T-cell acute lymphocytic leukemia with long disease latency.32, 33, 34, 35 However, the molecular mechanisms leading to disease and the cooperative nature of hyperactive Shp2 with leukemic fusion proteins has not been explored.
To investigate whether mutations associated with PTPN11 can cooperate with oncogenic fusion proteins, we developed a mouse model of cooperative leukemogenesis with MLL-AF9 and the leukemia-associated Shp2E76K mutant that shows the highest basal phosphatase activity among all the disease-associated Shp2 mutations.17, 36 Shp2E76K strongly cooperates with MLL-AF9 to accelerate leukemogenesis in mice by altering leukemic stem cell (LSC) frequency. MLL-AF9 Shp2E76K cells display cytokine hypersensitivity and activation of the Erk pathway, leading to upregulation of an anti-apoptotic gene program most prominently observed with Mcl1. We find that Shp2E76K expression in both mouse and human cells reduces MLL-AF9 sensitivity to chemical inhibition of Mcl1, suggesting mutant Shp2 cooperates mechanistically with MLL fusion proteins through Mcl1 expression.
Materials and methods
Female C57BL/6 mice at 8–10 weeks old were purchased from Taconic Farms (Germantown, NY, USA). B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J mice (TetOn mice) were purchased from Jackson laboratory (Bar Harbor, ME, USA). All animal studies were approved by the University of Michigan Committee on Use and Care of Animals and Unit for Laboratory Medicine.
Genetic analysis of primary patient AML samples
All primary patient samples came from pretreatment AML blood or bone marrow samples from patients treated on the ECOG E1900 clinical trial as previously described.37 All patient samples were previously evaluated for cytogenetic abnormalities and sequenced for the following genes recurrently mutated in AML: ASXL1, CEBPα, CKIT, DNMT3A, EZH2, FLT3, NPM1, HRAS, IDH1, IDH2, KRAS, NRAS, PHF6, WT1, RUNX1, TET2 and TP53.37 For sequencing of PTPN11, all coding regions of PTPN11 were amplified using RainDance microdroplet digital PCR enrichment (RainDance, Billerica, MA, USA) as previously described,38, 39 followed by Illumina HiSeq (Illumina, San Diego, CA, USA) massively parallel sequencing. Primer sequences are available on request.
MSCVpuro-Flag-mMcl1 plasmid was purchased from Addgene (Cambridge, MA, USA; 32982). MSCVneo-Flag-MLL-AF9 and MSCVneo-E2A-HLF (EH) plasmids have been described before.40 MIEG3 (retroviral vector using the bicistronic MSCV backbone and expressing enhanced green fluorescent protein) and MIEG3-Shp2E76K plasmids were provided by Dr Rebecca Chan (Indiana University).41 Short hairpin RNA (shRNA) plasmids were generated by inserting validated shRNA sequences into the XhoI and EcoRI sites of the pTRMPV-Hygro vector.42 Mcl1 shRNAs and Renilla control shRNA sequences were described previously.43 All shRNA sequences were listed in Supplementary Table 1.
Hematopoietic stem cells purified from TetOn mice were transformed with MLL-AF9 retrovirus. Freshly transformed (~2 weeks after transduction) cells were transduced with MIEG3 or MIEG3-Shp2E76K and sorted by green fluorescent protein expression. GFP+ cells were transduced with Plat-E packaged shRNA retroviruses. Cells were selected with hygromycin for 1 week (140 μg/ml for 5 days and 200 μg/ml for 2 days) with fresh antibiotics added every 1 to 2 days. shRNA-containing cells were mixed with shRNA-none cells (that is, TetOn MA9+MIEG3 or TetOn MA9+E76K) at 3:1 ratio and cultured in media containing 1 μg/ml Doxcycline. One day after Doxcycline treatment, cells were collected for flow cytometry to detect dsRed+ cell percentage in the mixed population. shRNA knockdown after doxycycline treatment were verified by western blotting (Germantown, NY, USA).
Chemical inhibition assay
All chemical inhibitors were dissolved in dimethyl sulfoxide. Cells were cultured at 5 × 104 cells/ml in 12-well non-tissue culture plate in the presence of serially diluted inhibitors. ABT-263 was added at 0.1, 0.33, 1, 2.5 and 5 μM. UMI-205, UMI-77, UMI-208 and UMI-212 were added at 1.25, 2.5, 5, 10 and 20 μM, respectively. Dimethyl sulfoxide was used as negative control. Cells were enumerated after 48 h of culture and the growth rate k was calculated according to the exponential growth equation:
PTPN11 mutations functionally cooperate with specific oncogenes in AML
To gain insight into the mutational frequency of SHP2 in human AML, we sequenced PTPN11 by next-generation sequencing in 91 AML patients enrolled in the phase III clinical trial run by ECOG E1900.47 Six of 91 patients were identified to contain a mutation in PTPN11 resulting in a 6.6% mutational frequency in AML, consistent with a saturation analysis of cancer genes in AML,20 suggesting PTPN11 mutations are functionally important to leukemic transformation (Table 1). All mutations were detected within exon 3 and resulted in amino acid changes previously associated with both AML and juvenile myelomonocytic leukemia.15, 17 The E76K, T31I, E76Q, D61Y, F71L and E76G mutations all reside within an auto-inhibitory region of the SHP2 protein and disrupts the N-SH2 interaction with the PTP domain, leading to hyperactive phosphatase activity.16, 28 PTPN11 mutations were found to co-occur with genetic alterations in DNMT3a, NPM1, WT1, CBF and MLL, suggesting mutant SHP2 may cooperate functionally in AML. As PTPN11 mutations were previously reported in MLL-associated leukemias,19 we tested whether hyperactive Shp2 can functionally cooperate with MLL fusion proteins in transformation. We performed primary bone marrow transduction and colony-replating assays, using retroviral vectors containing the oncogenic MSCV-MLL-AF9 (MA9) fusion protein and the hyperactive Shp2 mutant MIEG3-Shp2E76K (E76K) commonly associated with hematologic malignancies. Although expression of E76K alone did not lead to significant replating potential indicative of cellular transformation, the E76K mutant significantly augmented MLL-AF9-mediated colony formation as evidenced by a twofold increase in colony formation in the second round compared with MLL-AF9 and empty MIEG3 (Figures 1a and b). Colony formation between MA9 and MA9+E76K was similar in tertiary plating that may reflect more efficient transformation or limitations in quantifying colony-forming unit potential in the absence of limiting dilutions. MA9 and MA9+E76K colonies were dense and composed of immature blast-like cells with a greater nuclear to cytoplasmic ratio compared with the diffuse colonies composed of macrophage-type cells resulting from transduction with empty vectors or E76K alone (Figure 1b). Notably, although MLL-AF9 colonies were composed almost exclusively of primitive blast-like cells, MLL-AF9 and E76K co-transduced cells consistently contained sporadic differentiating macrophages (Figure 1b). To examine cooperation in other leukemic subtypes, we tested E76K expression in the presence of the unrelated leukemic oncoprotein EH that transforms cells through an anti-apoptotic gene program, which is distinct from the HOX gene program used by MLL fusion proteins.48 Interestingly, we did not observe a difference in EH-mediated colony-replating capacity in the presence of E76K (Figures 1a and b). Expression of Shp2E76K was confirmed by quantitative PCR using Shp2-specific primer sets and RNA isolated after the first round of the colony assay (Figure 1c). Together, these data identify PTPN11 mutations as co-occurring with several common hematologic genetic alterations and functionally cooperating with MLL-AF9-mediated transformation in vitro.
Hyperactive Shp2 accelerates MLL-AF9 leukemia and alters LSC frequency
We next assessed how Shp2 mutations affected MA9-mediated leukemogenesis in vivo. Here we introduced either empty vectors, MSCV+E76K, MA9+MIEG3 or MA9+E76K into lin−c-kit+ bone marrow cells by retroviral transduction. Cells were intravenously injected into lethally irradiated syngeneic recipients and allowed to engraft. Mice receiving cells transduced with empty vectors or MSCV+E76K did not show signs of disease through 150 days, suggesting overexpression of E76K alone failed to induce a lethal leukemia (Figure 2a). On the contrary, overexpression of E76K significantly accelerated MA9-mediated leukemogenesis, resulting in decreased disease latency (P=0.0004, log-rank test). The median survival of mice receiving MA9+MIEG3 cells (125 days) was more than double the median survival of mice receiving MA9+E76K (60 days; Figure 2a). Mice were monitored for 150 days with the first moribund MA9+E76K mouse euthanized on day 38 and the first MA9+MIEG3 mouse euthanized on day 103. MA9+MIEG3 or MA9+E76K mice displayed splenomegaly (Figure 2b) and compromised organ structure in the spleen and liver (Figure 2c). Histopathology revealed infiltrating leukemic blasts in the spleen and liver, while bone marrow aspirates revealed blocked myeloid differentiation in diseased MA9 and MA9+E76K mice (Figure 2c). Overexpression of Shp2E76K was confirmed by western blotting splenocytes from MA9 and MA9+E76K diseased mice (Figure 2d). It is noteworthy that MA9+E76K mice with longer disease latencies also showed the lowest expression of Shp2 by western blotting (Figure 2d). A correlation between Shp2E76K expression level and disease latency was established by plotting β-actin-normalized Shp2 protein expression levels against disease latency (R2=0.90367; Figure 2d). Overexpression of Ptpn11 was also confirmed by quantitative PCR using cDNA from diseased splenocytes (Figure 2e). Remarkably, we did not detect a difference in the expression of the direct MLL-AF9 target genes Hoxa9 and Meis1, suggesting the difference in disease latency is not due to a change in the MLL-AF9 gene program induced by Shp2E76K (Figure 2e). To follow up on the difference in cell morphology observed in colony assays with MA9 or MA9+E76K (Figure 1b), we performed flow cytometry on splenocytes. Cell surface expression of Sca1 and c-kit was similar in both MA9 and MA9+E76K cells (Figure 2f). However, expression of both Cd11b and Gr1 were significantly higher in MA9+E76K cells compared with that in MA9 cells, consistent with a slightly more differentiated phenotype (Figure 2f). These data are consistent with a role of Shp2 in promoting hematopoietic differentiation through dephosphorylation of Runx1.49 To understand whether accelerated leukemogenesis in the presence of E76K was due to altered LSC frequency, we performed a limiting dilution assay with primary leukemic cells isolated from MA9 and MA9+E76K diseased mice. These were injected into secondary recipients following sublethal irradiation and monitored for 109 days for disease. Although MA9 cells displayed an LSC frequency of ~1 in 285, the presence of E76K significantly increased LSC frequency to ~1 in 50 (Figure 2g). These data suggest Shp2E76K cooperates with the MLL-AF9 fusion protein in vivo to accelerate leukemogenesis by altering LSC frequency.
Shp2E76K affords MLL-AF9 cells’ cytokine hypersensitivity
To investigate the mechanism of accelerated leukemogenesis, we generated cell lines with MA9 or MA9+E76K by retroviral transduction of lin−c-kit+ bone marrow cells. We enriched for cells transduced with empty MIEG3 or E76K by sorting for green fluorescent protein, which revealed an expansion of bone marrow cells expressing E76K that was evident 4 days after transduction (Supplementary Figure 1). Proliferation assays demonstrated the E76K-mediated bone marrow expansion was transient, except in the presence of MLL-AF9, which initiated leukemic transformation (Figure 3a). Overexpression of mutant Shp2 was confirmed by western blotting (Figure 3b). Previous studies had implicated hyperactive Shp2 in hematopoietic progenitor cell cytokine hypersensitivity.31, 41 We tested this in the context of MLL-AF9 leukemic cells by performing an IL3 withdrawal and proliferation assay. We observed increased tolerance for IL3 withdrawal in MA9+E76K cells compared with that in MA9 (Figure 3c). This was also accompanied by increased Erk phosphorylation in the absence of IL3. MA9+E76K cells displayed higher resting levels of Erk phosphorylation with limiting amounts of IL3 and showed greater Erk phosphorylation in response to IL3 compared with MA9 cells (Figure 3d).
Several studies have linked Shp2 activity with the upregulation of anti-apoptotic genes.24, 27 Thus, we examined expression of both the Bcl2 family of genes and direct MLL fusion targets by quantitative PCR in MA9 and MA9+E76K cells, to reveal the mechanism for Shp2E76K-mediated accelerated leukemogenesis and cytokine hypersensitivity. We found the expression of Hoxa9 and Meis1 not significantly changed in the presence of Shp2E76K, suggesting modulation of MLL target genes is not responsible for cytokine hypersensitivity (Figure 4a). In contrast, we observed a significant upregulation of Bcl-xL and Mcl1 in MA9+E76K cells compared with MA9 cells in the presence of low-dose IL3 (0.001 ng/ml; Figure 4a). The most significant increase was observed in Mcl1 mRNA expression with a ~4-fold upregulation, which was confirmed by western blotting (Figure 4b). This is consistent with genetic analyses that identified Mcl1 as a key regulator of cell survival in MLL-ENL leukemic cells compared with Bcl2 or Bcl-xL.43 Interestingly, there were only minor changes in Hoxa9, Mcl1, Bcl2 or Bcl-xL expression when Shp2E76K was overexpressed in normal lin− bone marrow, suggesting a context-dependent function for hyperactive Shp2 (Supplementary Figure 2). Although all anti-apoptotic genes were upregulated in the presence of Shp2E76K, Mcl1 clearly emerged as the most highly expressed and upregulated in MA9+E76K cells (Figure 4a). These data suggest that the Shp2E76K mutation may accelerate leukemogenesis through the upregulation of an anti-apoptotic gene program primarily mediated by Mcl1.
Mcl1 expression recapitulates Shp2E76K-mediated cellular effects
As Mcl1 expression was notably higher than Bcl2 and Bcl-xL, we examined whether expression of Mcl1 alone would recapitulate the effects seen by Shp2E76K in MLL-AF9 cells. To this end, we used an MSCV-puro-based Mcl1 retroviral vector for co-transduction of lin− c-kit+ bone marrow cells along with MSCV-neo-MLL-AF9 and subsequent colony-forming and -replating assays. Similar to what was seen with co-transduction with Shp2E76K, a significant increase in colony formation was observed in the second round of the colony assay following co-transduction of MA9 and Mcl1 compared with MA9 and empty vector, and was evident by visual inspection of the plates (Figure 5a). Transduction with Mcl1 alone resulted in a similar number of colonies as empty vector-transduced cells and did not lead to replating capacity in the second and third round, suggesting Mcl1 overexpression alone is insufficient for transformation (Figure 5a). Stable cell lines were established from lin− c-kit+ bone marrow cells following transduction with MA9+MSCVpuro or MA9+Mcl1. To test the effect of Mcl1 expression on cytokine hypersensitivity, we tested proliferation rates in serially diluted concentrations of IL3. Overexpression of Mcl1 resulted in a marked proliferative advantage in lower doses of IL3 (Figure 5b). We next examined how Mcl1 expression affected colony-forming unit frequency, by performing a limiting dilution colony assay with MA9 and MA9+Mcl1. Strikingly, we observed a significantly higher frequency of colony-forming units per cell plated in MA9+Mcl1 (1/13.32) compared with MA9 alone (1/79.11; Figure 5c). Overexpression of Mcl1 was confirmed by western blotting in Figure 5d. Together, these data show expression of Mcl1 results in increased colony formation, cytokine hypersensitivity and increased colony-forming unit frequency, suggesting the cellular effects mediated by Shp2E76K may be mediated by expression of Mcl1.
AML cells with PTPN11 mutations are desensitized to Mcl1 inhibition
To further examine the role of anti-apoptotic proteins in MA9+E76K cells, we treated established mouse cell lines with chemical inhibitors with specificity towards different anti-apoptotic proteins. Here, the growth rate of MA9+MIEG3, MA9+E76K, MA9+Mcl1 and EH cells was assessed following treatment with varying concentrations of ABT-263, UMI-212 or UMI-205. ABT-263 is a cell-permeable BH3 mimetic chemical inhibitor that disrupts Bcl2 and Bcl-xL interactions with pro-death proteins such as Bim.50 UMI-212 is a novel cell-permeable chemical inhibitor that specifically targets Mcl1, without affecting Bcl2 or Bcl-xL, and UMI-205 is a control non-targeting compound.51, 52 As expected, no change in cell growth was observed in any cell lines following treatment of cells with UMI-205 (Figure 6a). In contrast, treatment of cells with ABT-263 resulted in decreased growth of EH cells without affecting the growth of MA9+MIEG3, MA9+E76K or MA9+Mcl1 cells (Figure 6a). EH cells showed an IC50 value of 1.80 μM compared with >5 μM for MA9+MIEG3, MA9+E76K and MA9+Mcl1 (Supplementary Table 3). These data suggest MLL fusion protein-transformed cells are less sensitive to Bcl2 inhibition compared with EH cells and are consistent with previous work implicating Bcl2 in EH-mediated leukemic transformation.48 On the contrary, MA9 cells were more sensitive to UMI-212-mediated Mcl1 inhibition compared with EH cells (Figure 6a). The sensitivity of MA9 cells to UMI-212 was reduced with the addition of E76K such that the growth pattern was similar to EH cells. These data are consistent with decreased cellular sensitivity to UMI-212 due to the upregulation of an anti-apoptotic program, most notably Mcl1, by E76K. In addition, direct overexpression of Mcl1 in MA9+Mcl1 cells led to less sensitivity to UMI-212, similar to expression of E76K (Figure 6a and Supplementary Table 3). MA9 cells have an IC50 of 5.197 μM compared with 8.389, 8.711 and 10.76 μM for EH, MA9+E76K and MA9+Mcl1 cells, respectively (Supplementary Table 3). Treatment with two additional chemical inhibitors with Mcl1 specificity, MI-77 and MI-228, yielded comparable results (Supplementary Figure 3). To test whether inhibition of Mcl1 led to programmed cell death, we examined cell viability by annexin V and 4',6-diamidino-2-phenylindole staining following treatment with control UMI-205, ABT-263 or UMI-212. Although UMI-205 did not affect cell viability, treatment with UMI-212 led to an increase in annexin V positivity indicative of programmed cell death (Figure 6b and Supplementary Figure 4). Although not statistically significant in MA9+E76K cells, a trend of decreased annexin V positivity was observed in MA9+E76K and MA9+Mcl1 cells compared with MA9 cells, consistent with the above growth curves and Mcl1 expression, suggesting leukemic cells with Shp2 mutations are less responsive to UMI-212-mediated Mcl1 inhibition (Figure 6a and b and Supplementary Figures 3 and 4). These data implicate Mcl1 as a crucial mediator of MLL-AF9 cell proliferation that is significantly modulated by Shp2E76K, rendering leukemic cells less sensitive to Mcl1 inhibition.
To verify the desensitization of MA9+E76K cells to UMI-212 was mediated by Mcl1 overexpression, we engineered inducible pTRMPV retroviral vectors to express validated shRNA targeting Mcl1 or control Renilla (Ren), to allow for gene-specific knockdown in leukemic cells. Freshly established TetOn MA9+MIEG3 and MA9+E76K cell lines were infected with the shRNA retroviruses and selected for 1 week. shRNA-containing cells were then mixed with parental cell lines lacking either pTRMPV vector at a 3:1 ratio and cultured with Doxycycline to induce shRNAs detectable by dsRed expression and flow cytometry. Consistent with previous data implicating Mcl1 gene expression in the survival of MLL leukemic cells,43 knockdown of Mcl1 with two separate shRNA vectors in MA9 cells led to a significantly reduced dsRed+ population in comparison with Ren controls (Figure 6c). Similarly, knockdown of Mcl1 in MA9+E76K cells lead to reduced dsRed+ population (Figure 6c). Knockdown of Mcl1 was confirmed by western blotting 24 h after Doxycycline treatment (Figure 6d).
We next tested how a variety of human AML cells respond to Mcl1 inhibition. To this end, we sequenced several human AML cell lines to determine the mutational status of exon 3 of PTPN11. Among the cell lines tested, only U937 cells contained a PTPN11 mutation (G60R) (Supplementary Figure 5). To determine how human leukemia cells containing a PTPN11 mutation would respond to anti-apoptotic protein inhibition, we treated the PTPN11 mutant U937 cell line and the PTPN11 wild-type cell lines K562, Monomac6, THP-1 and ML2 with ABT-263, UMI-212 or UMI-205. Although none of the cell lines showed a change in growth response to the control UMI-205 compound, U937 and THP1 cells were clearly more resistant to UMI-212-mediated Mcl1 inhibition compared with K562, MonoMac6 and ML2 cells (Figure 7a). U937 cells had an IC50 of 17.92 μM compared with IC50 values of 9.72 and 7.39 μM for K562 and ML2 cells, respectively, and 10.84 and 15.63 μM for MonoMac6 and THP1 cells, respectively (Supplementary Table 3). ML2 and THP1 cells showed slight sensitivity to ABT-263-mediated Bcl2/Bcl-xL inhibition (Figure 7a). These results were confirmed with additional Mcl1-specific inhibitors, which confirmed U937 desensitization to Mcl1 inhibition (Supplementary Figure 6). ALthough MCL1 expression varied between cell lines, colony-forming ability generally decreased in a dose-dependent manner with MCL1 inhibition (Supplementary Figure 7A and B). U937, K562 and THP1 cells displayed greater resistance to MCL1 inhibition compared with ML2 and MonoMac6 cells (Supplementary Figure 7A). The increased resistance of THP1 and K562 cells along with U937 cells in proliferation and colony assays likely reflect the utility of multiple transformation mechanisms that may compensate for loss of MCL1. Of note, THP1 cells harbor activating mutations in NRAS that may contribute to increased MCL1 expression. Despite these cell lines carrying a variety of driver mutations, including CALM-AF10, BCR-ABL, MLL-AF9 and MLL-AF6, these data suggest that a Shp2 mutation in human AML cells may result in greater resistance to MCL1 inhibition.
Recent deep-sequencing efforts have revealed a number of mutations that together with previously identified chromosomal abnormalities give a clearer picture of the genetic landscape of acute leukemias. The data presented currently provide an experimental validation of the premise proposed by Gilliland and Griffin10 that complete leukemogenesis require mutations conferring a block in differentiation and promoting cell survival. Although mouse models of both MLL-AF9 and Shp2E76K result in lethal leukemias,35, 53, 54 the long latencies of these diseases suggest cooperating events contribute to the disease. The presence of an MLL fusion protein, such as MLL-AF9, leads to a block in hematopoietic differentiation through the sustained expression of Hoxa9 and Meis1. The addition of PTPN11 gain-of-function mutations lead to functional cooperation between the MLL-AF9 fusion protein and mutated Shp2 that results in significantly accelerated leukemogenesis by increasing LSC frequency through upregulation of an anti-apoptotic gene program that primarily includes Mcl1 (Figure 7b). Upregulation of the Bcl2 family, namely Mcl1, induced by activating mutations in Shp2, leads to decreased sensitivity of both murine and human leukemia cell lines to Mcl1 inhibitors, suggesting that patients harboring PTPN11 mutations (or others within the Ras pathway) may show a poorer response to Mcl1 inhibition than patients without. Our data also suggest that the signaling pathways linking Shp2E76K and Mcl1 overexpression may represent candidates for therapeutic targeting in combination with Mcl1 inhibitors.
Mcl1 is associated with transformation and modulated by cooperating mutations
Elegant genetic studies have clearly demonstrated the importance of an anti-apoptotic gene program in AML cells using both overexpression and gene depletion techniques. For example, transgenic mice overexpressing Mcl1 in hematopoietic cells leads to increased survival in several hematopoietic lineages and immortalization of myeloid cells in the presence of IL3 ex vivo.55 Further, mice expressing a modified allele of Mcl1 that encodes an abnormally stabilized form of Mcl1 showed accelerated AML induced by overexpression of c-MYC.56 These studies are important, as MCL1 was shown to be the predominant Bcl2 family member overexpressed in AML patient samples. Using conditional knock out alleles, Glaser et al.43 demonstrated the importance of Mcl1, Bcl2 and Bcl-xL in AML cell survival. Interestingly, it was shown that MLL fusion protein-mediated leukemogenesis was most severely inhibited by deletion of Mcl1. However, AML cell death following loss of Mcl1 can be compensated for by expression of Bcl2. Further, cell death induced by loss of Mcl1 is augmented by treatment with ABT-737, suggesting non-overlapping and cooperative roles for Mcl1 and Bcl2 or Bcl-xL in AML cell survival.43 Together, these data are consistent with our gene expression analysis showing Mcl1 as the most highly expressed of the anti-apoptotic genes in MLL-AF9+Shp2E76K cells (Figure 4). Further, we observed differential sensitivity between MLL-AF9 and EH AML cells following treatment with the Bcl2/Bcl-xL inhibitor ABT-263 and a Mcl1 inhibitor (Figure 6). EH cells showed more sensitivity to Bcl2 inhibition compared with MLL-AF9 cells; however, MLL-AF9 cells are more sensitive to Mcl1 inhibition compared with EH cells. This is consistent with the finding that EH cells transform through the induced expression of Bcl2, whereas MLL fusion cells are more dependent on Mcl1.43, 48
Our data suggest a strong link between Mcl1 expression, mediated by Shp2, and increased LSC frequency. Among the anti-apoptotic family of proteins, Mcl1 is indispensible for the self-renewal of normal hematopoietic stem cells.57 These data may suggest similar mechanisms governing the self-renewal of both normal hematopoietic stem cells and LSCs that may reflect an obstacle to therapeutic targeting. Indeed, upregulation of Mcl1 and Bcl-xL by Shp2 was reported in normal hematopoietic stem cells and regulation of Bcl-xL by Shp2 was reported in FLT3-ITD-positive leukemic cells.24, 27 Further, differential regulation of Bcl-xL was reported by Shp2 in human leukemic HL60 cells compared with MV4;11 cells.27 Thus, Shp2 may differentially regulate anti-apoptotic genes dependent on the leukemic subtype. Further studies characterizing anti-apoptotic gene expression in various leukemic subtypes will be required to better understand this regulation.
Raimondi SC, Chang MN, Ravindranath Y, Behm FG, Gresik MV, Steuber CP et al. Chromosomal abnormalities in 478 children with acute myeloid leukemia: clinical characteristics and treatment outcome in a cooperative pediatric oncology group study-POG 8821. Blood 1999; 94: 3707–3716.
Rubnitz JE, Link MP, Shuster JJ, Carroll AJ, Hakami N, Frankel LS et al. Frequency and prognostic significance of HRX rearrangements in infant acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood 1994; 84: 570–573.
Huret JL, Minor SL, Dorkeld F, Dessen P, Bernheim A . Atlas of genetics and cytogenetics in oncology and haematology, an interactive database. Nucleic Acids Res 2000; 28: 349–351.
Bitoun E, Oliver PL, Davies KE . The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling. Hum Mol Genet 2007; 16: 92–106.
Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L et al. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell 2010; 37: 429–437.
Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML . A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell 2010; 17: 198–212.
Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden MD et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet 2002; 30: 41–47.
Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 2007; 446: 758–764.
Balgobind BV, Zwaan CM, Pieters R, Van den Heuvel-Eibrink MM . The heterogeneity of pediatric MLL-rearranged acute myeloid leukemia. Leukemia 2011; 25: 1239–1248.
Gilliland DG, Griffin JD . The roles of FLT3 in hematopoiesis and leukemia. Blood 2002; 100: 1532–1542.
Ono R, Nakajima H, Ozaki K, Kumagai H, Kawashima T, Taki T et al. Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis. J Clin Invest 2005; 115: 919–929.
Stubbs MC, Kim YM, Krivtsov AV, Wright RD, Feng Z, Agarwal J et al. MLL-AF9 and FLT3 cooperation in acute myelogenous leukemia: development of a model for rapid therapeutic assessment. Leukemia 2008; 22: 66–77.
Kim WI, Matise I, Diers MD, Largaespada DA .. RAS oncogene suppression induces apoptosis followed by more differentiated and less myelosuppressive disease upon relapse of acute myeloid leukemia. Blood 2009; 113: 1086–1096.
Mohi MG, Neel BG . The role of Shp2 (PTPN11) in cancer. Curr Opin Genet Dev 2007; 17: 23–30.
Chan G, Kalaitzidis D, Neel BG . The tyrosine phosphatase Shp2 (PTPN11) in cancer. Cancer Meta Rev 2008; 27: 179–192.
Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001; 29: 465–468.
Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003; 34: 148–150.
Paulsson K, Horvat A, Strombeck B, Nilsson F, Heldrup J, Behrendtz M et al. Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer 2008; 47: 26–33.
Tartaglia M, Martinelli S, Cazzaniga G, Cordeddu V, Iavarone I, Spinelli M et al. Genetic evidence for lineage-related and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia. Blood 2004; 104: 307–313.
Lawrence MS, Stojanov P, Mermel CH, Robinson JT, Garraway LA, Golub TR et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 2014; 505: 495–501.
Welch JS, Ley TJ, Link DC, Miller CA, Larson DE, Koboldt DC et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 2012; 150: 264–278.
Neel BG, Gu H, Pao L . The 'Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem Sci 2003; 28: 284–293.
Zhu HH, Ji K, Alderson N, He Z, Li S, Liu W et al. Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool. Blood 2011; 117: 5350–5361.
Li L, Modi H, McDonald T, Rossi J, Yee JK, Bhatia R .. A critical role for SHP2 in STAT5 activation and growth factor-mediated proliferation, survival, and differentiation of human CD34+ cells. Blood 2011; 118: 1504–1515.
Chan G, Cheung LS, Yang W, Milyavsky M, Sanders AD, Gu S et al. Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells. Blood 2011; 117: 4253–4261.
Xu R, Yu Y, Zheng S, Zhao X, Dong Q, He Z et al. Overexpression of Shp2 tyrosine phosphatase is implicated in leukemogenesis in adult human leukemia. Blood 2005; 106: 3142–3149.
Nabinger SC, Li XJ, Ramdas B, He Y, Zhang X, Zeng L et al. The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo. Leukemia 2013; 27: 398–408.
Hof P, Pluskey S, Dhe-Paganon S, Eck MJ, Shoelson SE . Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998; 92: 441–450.
Stein-Gerlach M, Wallasch C, SHP-2 Ullrich A .. SH2-containing protein tyrosine phosphatase-2. Int J Biochem Cell Biol 1998; 30: 559–566.
Barford D, Neel BG . Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2. Structure 1998; 6: 249–254.
Schubbert S, Lieuw K, Rowe SL, Lee CM, Li X, Loh ML et al. Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells. Blood 2005; 106: 311–317.
Xu D, Liu X, Yu WM, Meyerson HJ, Guo C, Gerson SL et al. Non-lineage/stage-restricted effects of a gain-of-function mutation in tyrosine phosphatase Ptpn11 (Shp2) on malignant transformation of hematopoietic cells. J Exp Med 2011; 208: 1977–1988.
Chan G, Kalaitzidis D, Usenko T, Kutok JL, Yang W, Mohi MG et al. Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis. Blood 2009; 113: 4414–4424.
Mohi MG, Williams IR, Dearolf CR, Chan G, Kutok JL, Cohen S et al. Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 2005; 7: 179–191.
Xu D, Wang S, Yu WM, Chan G, Araki T, Bunting KD et al. A germline gain-of-function mutation in Ptpn11 (Shp-2) phosphatase induces myeloproliferative disease by aberrant activation of hematopoietic stem cells. Blood 2010; 116: 3611–3621.
Yu ZH, Xu J, Walls CD, Chen L, Zhang S, Zhang R et al. Structural and mechanistic insights into LEOPARD syndrome-associated SHP2 mutations. J Biol Chem 2013; 288: 10472–10482.
Patel JP, Gonen M, Figueroa ME, Fernandez H, Sun Z, Racevskis J et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med 2012; 366: 1079–1089.
ElSharawy A, Warner J, Olson J, Forster M, Schilhabel MB, Link DR et al. Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing. BMC Genomics 2012; 13: 500.
Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 2009; 27: 1025–1031.
Muntean AG, Chen W, Jones M, Granowicz EM, Maillard I, Hess JL . MLL fusion protein-driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction. Blood 2013, 30.
Chan RJ, Leedy MB, Munugalavadla V, Voorhorst CS, Li Y, Yu M et al. Human somatic PTPN11 mutations induce hematopoietic-cell hypersensitivity to granulocyte-macrophage colony-stimulating factor. Blood 2005; 105: 3737–3742.
Zuber J, McJunkin K, Fellmann C, Dow LE, Taylor MJ, Hannon GJ et al. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol 2011; 29: 79–83.
Glaser SP, Lee EF, Trounson E, Bouillet P, Wei A, Fairlie WD et al. Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia. Genes Dev 2012; 26: 120–125.
Muntean AG, Giannola D, Udager AM, Hess JL . The PHD fingers of MLL block MLL fusion protein-mediated transformation. Blood 2008; 112: 4690–4693.
Tan J, Jones M, Koseki H, Nakayama M, Muntean AG, Maillard I et al. CBX8, a polycomb group protein, is essential for MLL-AF9-induced leukemogenesis. Cancer Cell 2011; 20: 563–575.
Hu Y, Smyth GK . ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 2009; 347: 70–78.
Fernandez HF, Sun Z, Yao X, Litzow MR, Luger SM, Paietta EM et al. Anthracycline dose intensification in acute myeloid leukemia. N Engl J Med 2009; 361: 1249–1259.
Inaba T, Inukai T, Yoshihara T, Seyschab H, Ashmun RA, Canman CE et al. Reversal of apoptosis by the leukaemia-associated E2A-HLF chimaeric transcription factor. Nature 1996; 382: 541–544.
Huang H, Woo AJ, Waldon Z, Schindler Y, Moran TB, Zhu HH et al. A Src family kinase-Shp2 axis controls RUNX1 activity in megakaryocyte and T-lymphocyte differentiation. Genes Dev 2012; 26: 1587–1601.
Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 2008; 68: 3421–3428.
Abulwerdi F, Liao C, Liu M, Azmi AS, Aboukameel A, Mady AS et al. A novel small-molecule inhibitor of mcl-1 blocks pancreatic cancer growth in vitro and in vivo. Mol Cancer Ther 2014; 13: 565–575.
Abulwerdi FA, Liao C, Mady AS, Gavin J, Shen C, Cierpicki T et al. 3-substituted-N-(4-hydroxynaphthalen-1-yl)arylsulfonamides as a novel class of selective Mcl-1 inhibitors: structure-based design, synthesis, SAR, and biological evaluation. J Med Chem 2014; 57: 4111–4133.
Dobson CL, Warren AJ, Pannell R, Forster A, Lavenir I, Corral J et al. The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis. EMBO J 1999; 18: 3564–3574.
Lavau C, Szilvassy SJ, Slany R, Cleary ML . Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J 1997; 16: 4226–4237.
Zhou P, Qian L, Bieszczad CK, Noelle R, Binder M, Levy NB et al. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 1998; 92: 3226–3239.
Okamoto T, Coultas L, Metcalf D, van Delft MF, Glaser SP, Takiguchi M et al. Enhanced stability of Mcl1, a prosurvival Bcl2 relative, blunts stress-induced apoptosis, causes male sterility, and promotes tumorigenesis. Proc Natl Acad Sci USA 2014; 111: 261–266.
Campbell CJ, Lee JB, Levadoux-Martin M, Wynder T, Xenocostas A, Leber B et al. The human stem cell hierarchy is defined by a functional dependence on Mcl-1 for self-renewal capacity. Blood 2010; 116: 1433–1442.
We thank Dr Jay Hess for helpful discussion. This work was supported by NIH grants R01-CA149442 (ZN-C), R00 CA158136 (AGM) and an American Society of Hematology Scholar Award (AGM)
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
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Chen, L., Chen, W., Mysliwski, M. et al. Mutated Ptpn11 alters leukemic stem cell frequency and reduces the sensitivity of acute myeloid leukemia cells to Mcl1 inhibition. Leukemia 29, 1290–1300 (2015). https://doi.org/10.1038/leu.2015.18
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