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Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R–targeted antibodies. Sensitivity to the IGF-1R–targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1–resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R–targeted antibodies tested against this model.

Purpose: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. Experimental Design: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Supplemental data

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF–MEK–ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF–MEK–ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R–targeted antibodies. Sensitivity to the IGF-1R–targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1–resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R–targeted antibodies tested against this model.

Supplementary Materials, Methods, and References

Supplementary Methods

Supplementary figures and legends

Supplementary Figures and Tables

Pharmacodynamic response to trametinib/ganitumab in xenograft models does not predict tumor shrinkage

Related to Figure 4

Purpose:PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS.Experimental Design:Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models.Results:Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis.Conclusions:We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Sample Sizes

Related to Figure 1B

The combination of trametinib and ganitumab does not induce hyperinsulinemia or hematologic adverse events in SCID beige mice

Effect of trametinib and ganitumab in additional cell culture models of RAS-mutated RMS

Copy Number

Related to Figure 2 - SMS-CTR

Related to Figure 3

Mechanism of decreased PTEN expression in RD

Related to Figure 1D

Schematic depicting the impact of PTEN loss on sensitivity to MEK inhibition

Combining ganitumab with trametinib inhibits tumor growth and increases fibrosis in SMS-CTR but not RD xenografts

Related to Figure 2 - RD

Baseline Statistics

Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.

Materials and Methods used for experiments in Supplemental Figures and Supplemental References

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy.Significance:SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

Supplementary Figures S1-S9

Supplemental Tables 1-5

Supplementary Data from Radiosensitization of Epidermal Growth Factor Receptor/HER2–Positive Pancreatic Cancer Is Mediated by Inhibition of Akt Independent of Ras Mutational Status

Purpose: Epidermal growth factor receptor (EGFR) family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations.Experimental Design: Expression of EGFR family members in pancreatic cancer lines was assessed by quantitative reverse transcription-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity.Results: Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras–expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was seen in lines expressing mutant K-ras. Overexpression of constitutively active K-ras (G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MAP/ERK kinase/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio, 1.2-1.8) or nelfinavir (enhancement ratio, 1.2-1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras–containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5).Conclusions: Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other phosphoinositide 3-kinase/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status. Clin Cancer Res; 16(3); 912–23

Supplementary Figures 1-9. Figure S1: Auto-phosphorylation of EGFR-A289V and EGFR-G598V can be inhibited by EGFR TKIs. Figure S2: EGFR* expression is strongly induced in hGFAP-tTA tetO-EGFR* off-Dox mouse brains. Figure S3: Gfap and Nestin protein expression are overlapped in a subpopulation of NPCs. Figure S4: Representative H&E images of a Grade IV malignant glioma. Figure S5: Malignant glioma cells do not express terminally differentiated CNS cell lineage markers. Figure S6: iEIP glioma subcutaneous transplants are sensitive to genetic suppression of EGFR* induction but not to EGFR TKI. Figure S7: iEIP gliomas are sensitive to acute EGFR* ablation. Figure S8: Ingenuity pathway analysis comparing gene expression profiling of untreated control tumors with Dox-treated relapsed tumors. Figure S9: Combined treatment of Dox and Bez-235 inhibits EGFR* induction and Akt activation.

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF–MEK–ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF–MEK–ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. Significance: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

In fusion-negative rhabdomyosarcoma (FN-RMS), a pediatric malignancy with skeletal muscle characteristics, >90% of high-risk patients have mutations that activate the RAS/MEK signaling pathway. We recently discovered that SNAI2, in addition to blocking myogenic differentiation downstream of MEK signaling in FN-RMS, represses proapoptotic BIM expression to protect RMS tumors from ionizing radiation (IR). As clinically relevant concentrations of the MEK inhibitor trametinib elicit poor responses in preclinical xenograft models, we investigated the utility of low-dose trametinib in combination with IR for the treatment of RAS-mutant FN-RMS. We hypothesized that trametinib would sensitize FN-RMS to IR through its downregulation of SNAI2 expression. While we observed little to no difference in myogenic differentiation or cell survival with trametinib treatment alone, robust differentiation and reduced survival were observed after IR. In addition, IR-induced apoptosis was significantly increased in FN-RMS cells treated concurrently with trametinib, as was increased BIM expression. SNAI2’s role in these processes was established using overexpression rescue experiments, where overexpression of SNAI2 prevented IR-induced myogenic differentiation and apoptosis. Moreover, combining MEK inhibitor with IR resulted in complete tumor regression and a 2- to 4-week delay in event-free survival (EFS) in preclinical xenograft and patient-derived xenograft models. Our findings demonstrate that the combination of MEK inhibition and IR results in robust differentiation and apoptosis, due to the reduction of SNAI2, which leads to extended EFS in FN-RMS. SNAI2 thus is a potential biomarker of IR insensitivity and target for future therapies to sensitize aggressive sarcomas to IR.

Aim: Despite aggressive multiagent protocols, patients with metastatic rhabdomyosarcoma (RMS) have poor prognosis. In a recent high-risk trial (ARST0431), 25% of patients failed within the first year, while on therapy and 80% had tumor progression within 24 months. However, the mechanisms for tumor resistance are essentially unknown. Here we explore the use of preclinical models to develop resistance to complex chemotherapy regimens used in ARST0431. Methods: A Single Mouse Testing (SMT) protocol was used to evaluate the sensitivity of 34 RMS xenograft models to one cycle of vincristine, actinomycin D, cyclophosphamide (VAC) treatment. Tumor response was determined by caliper measurement, and tumor regression and event-free survival (EFS) were used as endpoints for evaluation. Treated tumors at regrowth were transplanted into recipient mice, and the treatment was repeated until tumors progressed during the treatment period (i.e., became resistant). At transplant, tumor tissue was stored for biochemical and omics analysis. Results: The sensitivity to VAC of 34 RMS models was determined. EFS varied from 3 weeks to > 20 weeks. Tumor models were classified as having intrinsic resistance, intermediate sensitivity, or high sensitivity to VAC therapy. Resistance to VAC was developed in multiple models after 2-5 cycles of therapy; however, there were examples where sensitivity remained unchanged after 3 cycles of treatment. Conclusion: The SMT approach allows for in vivo assessment of drug sensitivity and development of drug resistance in a large number of RMS models. As such, it provides a platform for assessing in vivo drug resistance mechanisms at a “population” level, simulating conditions in vivo that lead to clinical resistance. These VAC-resistant models represent “high-risk” tumors that mimic a preclinical phase 2 population and will be valuable for identifying novel agents active against VAC-resistant disease.

Purpose of review The current review aims to highlight the frequency of RAS mutations in pediatric leukemias and solid tumors and to propose strategies for targeting oncogenic RAS in pediatric cancers. Recent findings The three RAS genes (HRAS, NRAS, and KRAS) comprise the most frequently mutated oncogene family in human cancer. RAS mutations are commonly observed in three of the leading causes of cancer death in the United States, namely lung cancer, pancreatic cancer, and colorectal cancer. The association of RAS mutations with these aggressive malignancies inspired the creation of the National Cancer Institute RAS initiative and spurred intense efforts to develop strategies to inhibit oncogenic RAS, with much recent success. RAS mutations are frequently observed in pediatric cancers; however, recent advances in anti-RAS drug development have yet to translate into pediatric clinical trials. Summary We find that RAS is mutated in common and rare pediatric malignancies and that oncogenic RAS confers a functional dependency in these cancers. Many strategies for targeting RAS are being pursued for malignancies that primarily affect adults and there is a clear need for inclusion of pediatric patients in clinical trials of these agents.

Background: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS75807, but this combination was not pursued clinically due to excessive toxicity in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor that would be better tolerated in murine models and effective in both cell line and patient derived xenograft models of RAS-mutant FN RMS. Methods: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a monoclonal antibody with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of the combination was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in five out of six models of RAS-mutant RMS. Evidence suggests that the combination had little effect on body weight loss, thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the AKT phosphorylation that is induced by MEK inhibition alone. Therapeutic response to the combination was observed in models with an intact PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. The trametinib/ganitumab combination also likely has an improved tolerability profile compared to other IGF1R/MEK inhibitor combinations. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS. Citation Format: Marielle E. Yohe, Katie E. Hebron, Xiaolin Wan, Jacob S. Roth, David J. Liewehr, Nancy E. Sealover, Stacey Stauffer, Olivia Feehan-Nelson, Wenyue Sun, Kristine A. Isanogle, Christina M. Robinson, Amy James, Parirokh Awasthi, Priya Shankarappa, Xiaoling Liu, Haiyan Lei, Donna Butcher, Roberta Smith, Elijah F. Edmonson, Jin-Qui Chen, Noemi Kedei, Cody S. Peer, Jack F. Shern, W. Douglas Figg, Lu Chen, Matthew D. Hall, Simone Difillipantonio, Frederic G. Barr, Robert L. Kortum, Angelina V. Vaseva, Javed Khan. Therapeutic efficacy of trametinib and ganitumab in RAS-mutated rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA023.

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X-mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X-dependency in RMS cells, however there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade, and particularly co-targeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X-mutant RMS tumor growth.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma (RMS), yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple RMS cell lines. Modulating SNAI2 levels in RMS cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and ChIP-seq experiments established that SNAI2 is a direct repressor of BIM. Since the p53 pathway is nonfunctional in the RMS cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve RMS therapy.

Rhabdomyosarcoma (RMS) is the most commonly diagnosed soft tissue sarcoma in children. While the 5-year event-free survival for localized rhabdomyosarcoma is over 70%, it is less than 30% for patients presenting with metastatic disease. Given that the current treatment modalities for RMS patients remain cytotoxic therapies with profound life-long lasting complications, identification of less toxic and more effective therapies is highly desired. Whole-genome and RNA sequencing of human rhabdomyosarcoma have identified frequent RAS genes oncogenic mutations in fusion-negative rhabdomyosarcoma (FN-RMS), which comprises 65% of RMS. This high incidence of RAS mutations present opportunity for design of targeted therapeutic strategies for FN-RMS. By siRNA-mediated knock down of individual RAS isoforms we showed that H/NRAS Q61X-mutant FN-RMS cell lines exhibited oncogenic RAS dependency and that knockdown of the Q61X-mutant H/NRAS inhibited ERK MAPK, but not PI3K-AKT pathway activity. Knockdown of the Q61X-mutant H/NRAS phenocopied ERK inhibitor treatment by inducing myogenic differentiation. Further, as compared to RAS-wild type RMS cells, H/NRAS-Q61X FN-RMS cell lines were preferentially more sensitive to MEK or ERK, but not PI3K or AKT inhibitors. RNA sequencing analysis revealed significant overlap of genes signatures associated with NRAS-Q61H knockdown and ERK inhibitor treatment in the FN-RMS cell line RD. These results indicated a central role of the ERK MAPK pathway in mediating H/NRAS Q61X-dependency in FN-RMS. However, in vivo evaluation of clinically relevant exposers with the MEK inhibitor trametinib demonstrated poor response, which correlated with inefficient ERK inhibition in pharmacodynamic assays. A trametinib-sensitizing CRISPR screen pointed to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically blocked ERK activity and induced cell cycle arrest, myogenic differentiation and cell death. In vivo evaluation of low dose pan-RAFi + ERKi or MEKi + ERKi potently suppressed H/NRAS Q61X RMS tumor growth, with pan-RAFi + ERKi being more effective and better tolerated, suggesting wider therapeutic window. These results support the clinical translation of pan-RAFi + MEKi/ERKi for H/NRAS Q61X-mutant FN-RMS. Citation Format: Natalia Garcia, Vanessa Del-Pozo, Marielle Yohe, Craig Goodwin, Terry Shackleford, Long Wang, Kunal Baxi, Yidong Chen, Myron Ignatius, Kris Wood, Peter Houghton, Angelina Vaseva. Vertical inhibition of the RAF MEK ERK cascade induces myogenic differentiation, apoptosis and tumor regression in H/NRAS Q61X mutant rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB251.

Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry-based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62 Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.

RAS pathway mutations are found in nearly 75% of high-risk embryonal rhabdomyosarcoma (ERMS). While RAS oncoproteins are well-established therapeutic targets for many adult human cancers, still very little is known about the role of RAS mutations in the development and maintenance of ERMS. By sequencing, we identified cell lines and PDX tumors harboring activating mutations in H- or NRAS. Further, we showed that mutant H- or NRAS was critical for the growth of all RAS-mutant ERMS cell lines and that RAF/MEK/ERK signaling pathway, but not PI3K/AKT, was mediator of RAS dependency in these cells. However, in vivo treatment of RAS-mutant ERMS xenografts with the MEK inhibitor trametinib showed modest response as compared to BRAF-mutant astrocytoma xenografts. We reasoned that similarly to other RAS-driven cancers, ERMS cells and tumors are able to acquire resistance to inhibitors of the RAF/MEK/ERK pathway. We performed drug-sensitizing pooled CRISPR library screen and identified that inhibition of ERK2 potentiated trametinib treatment. We show that combining trametinib with ERK1/2 inhibitor leads to potent synergistic ERK inhibition and ERMS tumor growth suppression. Citation Format: Angelina V. Vaseva, Abhik Bandyopadhyay, Vanessa Del Pozo, Craig M. Goodwin, Prson Gautam, Krister Wennerberg, Kris C. Wood, Yidong Chen, Channing J. Der, Peter J. Houghton. Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A54.

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from "Rasless" cells expressing the oncoprotein BRAFV600E Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.

Our recent ERK inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) demonstrated that MYC protein loss is a marker of inhibitor sensitivity, and indicated that resistant cell lines possess KRASdependent, ERK-independent mechanisms maintaining MYC protein levels. In seeking to identify these mechanisms, we first determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, this MYC degradation was independent of both PI3K-AKTGSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation, and performed a kinome-wide proteomics screen. We identified an ERK1/2- inhibition-induced feed-forward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing MYC degradation. We further determined that concurrent inhibition of ERK1/2 and ERK5 disrupted this feed-forward compensatory mechanism, synergistically causing loss of MYC and suppressing PDAC growth.

SUMMARY Ischemia-associated oxidative damage leading to necrosis is a major cause of catastrophic tissue loss, and elucidating its signaling mechanism is therefore of paramount importance. p53 is a central stress sensor responding to multiple insults, including oxidative stress to orchestrate apoptotic and autophagic cell death. Whether p53 can also activate oxidative stress-induced necrosis is, however, unknown. Here, we uncover a role for p53 in activating necrosis. In response to oxidative stress, p53 accumulates in the mitochondrial matrix and triggers mitochondrial permeability transition pore (PTP) opening and necrosis by physical interaction with the PTP regulator cyclophilin D (CypD). Intriguingly, a robust p53-CypD complex forms during brain ischemia/reperfusion injury. In contrast, reduction of p53 levels or cyclosporine A pretreatment of mice prevents this complex and is associated with effective stroke protection. Our study identifies the mitochondrial p53-CypD axis as an important contributor to oxidative stress-induced necrosis and implicates this axis in stroke pathology.