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

Ageing is a phenomenon in which cells, tissues and organs undergo systemic pathological changes as individuals age, leading to the occurrence of ageing-related diseases and the end of life. It is associated with many phenotypes known as ageing characteristics, such as genomic instability, nutritional imbalance, mitochondrial dysfunction, cell senescence, stem cell depletion, and an altered microenvironment. The sirtuin family (SIRT), known as longevity proteins, is thought to delay ageing and prolong life, and mammals, including humans, have seven family members (SIRT1-7). SIRT4 has been studied less among the sirtuin family thus far, but it has been reported that it has important physiological functions in organisms, such as promoting DNA damage repair, participating in the energy metabolism of three substances, inhibiting inflammatory reactions and apoptosis, and regulating mitochondrial function. Recently, some studies have demonstrated the involvement of SIRT4 in age-related processes, but knowledge in this field is still scarce. Therefore, this review aims to analyse the relationship between SIRT4 and ageing characteristics as well as some age-related diseases (e.g., cardiovascular diseases, metabolic diseases, neurodegenerative diseases and cancer).

Abstract 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.

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.

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.

Abstract 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.

Abstract Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS.

Background: Several pediatric solid tumors, such as rhabdomyosarcoma (RMS) and neuroblastoma (NB) are driven by alterations in the RAS/MAP kinase pathway and are partially responsive to MEK inhibition. Overexpression of the IGF1R as well as its ligands has been observed in multiple malignancies, including pediatric sarcomas and NB. Preclinical and clinical studies have suggested that IGF1R is itself an important target in these diseases. Previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an inhibitor of IGF1R, BMS75807, in cell line xenograft models of RAS-mutated RMS; however, clinical translation of this combination was limited by toxicity. Here, we sought to identify a combination of a MEK1/2 inhibitor and IGF1R inhibitor that would be better tolerated.

To determine what alternative pathways may act as mechanisms of bypass resistance to type 1 insulin-like growth factor receptor (IGF-1R) blockade in rhabdomyosarcoma (RMS), we compared expression of receptor tyrosine kinase activity in a number of IGF-1R antibody-resistant and -sensitive RMS cell lines. We found that platelet-derived growth factor receptor β (PDGFR-β) activity was upregulated in three xenograft-derived IGF-1R antibody-resistant cell lines that arose from a highly sensitive fusion-positive RMS cell line (Rh41). Furthermore, we identified four additional fusion-negative RMS cell lines that similarly upregulated PDGFR-β activity when selected for IGF-1R antibody resistance in vitro. In the seven cell lines described, we observed enhanced growth inhibition when cells were treated with dual IGF-1R and PDGFR-β inhibition in vitro. In vivo studies have confirmed the enhanced effect of targeting IGF-1R and PDGFR-β in several mouse xenograft models of fusion-negative RMS. These findings suggest that PDGFR-β acts as a bypass resistance pathway to IGF-1R inhibition in a subset of RMS. Therapy co-targeting these receptors may be a promising new strategy in RMS care.

The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling.However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic.In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR).We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS).Combining anti-IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition-induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo.Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo.This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS.Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition.Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.

10046 Background: IGF-1R and its ligands have been shown to be potentially important therapeutic targets for sarcomas. Phase II sarcoma trials using IGF-1R blockade yielded clinically meaningful responses in unselected patients with pediatric type sarcomas. However, most responses were short-lived with rapid onset of resistance. Similarly, data from mouse xenograft RMS models showed initial response with subsequent resistance. Evaluation of tumor samples from treated mice showed persistently down-regulated IGF-1R, but rebound AKT phosphorylation, suggesting that resistance was not from loss of antibody activity against IGF-1R, but rather the result of a bypass pathway. We therefore sought to model acquired resistance in human RMS cells. Methods: Human RMS cell lines resistant to IGF-1R blockade were generated in xenografts from a highly sensitive parent cell line. Additional cell lines were selected for in vitro resistance. Parental and resistant lines were screened with a receptor phosphotyrosine array. Differences in expression were confirmed with Western Blot analysis and electrochemiluminescence detection. Cell growth under dual pathway inhibition was tested in vitro using IGF-1R antibody and several kinase inhibitors. Results: Screening of parental and resistant lines revealed increased phosphorylation of PDGFR-β in resistant cells. PDGFR-β and IGF-1R share several signaling pathways (PI3K, MAPK/ERK), suggesting that PDGFR-β activation may act as a bypass mechanism to activate AKT. Target inhibition of PDGFR-β in resistant cells was achieved with pazopanib. Proliferation assays have shown that dual inhibition of IGF-1R and PDGFR-β in resistant clones enhances cell growth inhibition in vitro. Similar studies are ongoing using xenografted tumors. Conclusions: Activation of PDGFR-β may be responsible for resistance to IGF-1R blockade in some RMS. This mechanism may also explain the rapid onset of resistance seen in clinical trials using IGF-1R blockade. Therapy targeting both IGF-1R and PDGFR-β may be a rational combination to treat RMS and prevent onset of resistance in some patients.

10045 Background: The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. Thus, identifying the mechanisms of acquired resistance to IGF-1R blockade is a major goal. The aim of this study was to identify the molecular mechanisms responsible for acquired resistance to IGF-1R targeted therapy in rhabdomyosarcoma. Methods: Expression profiles of IGF components and Src family kinases (SFKs) were analyzed by cDNA microarray. Antiproliferative effects of anti-IGF-1R agents and SFK inhibitors alone or in combination were tested in vitro in multiple rhabdomyosarcoma (RMS) cell lines and in vivousing xenografts. Western blot and immunoprecipitation were performed to identify protential resistance mechanisms to IGF-1R inhibition. Results: We identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor against IGF-1R/IR. In both cases, resistance was associated with increased activation of Yes/SFK in RMS. Combining anti-IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition induced activation of Yes/SFK and displayed enhanced antitumor activity in vitro and in vivo. Conclusions: Our data provide evidence that IGF-1R blockade results in activation of the Yes/Src family kinases (SFKs) by-pass resistance pathway in vitro and in vivo, and that co-targeting both IGF-IR and SFK shows advantageous antitumor activity in vitro and in vivo. Our preclinical data support consideration of clinical trials to test this combination in pediatric sarcomas.

Abstract Our group, and many others have documented a crucial role for IGFIR signaling in several pediatric sarcomas. These studies, along with the development of fully human IGFIR antibodies led to clinical testing of these blocking antibodies in rhabdomyosarcoma, Ewing's sarcoma, and osteosarcoma, along with a variety of other sarcomas. In general these single agent Phase 2 studies showed objective responses rates below 20%. Furthermore, even in responding patients, the duration of response is short-lived, typically less than 18 weeks. Thus the majority of patients do not have long term benefit from IGFIR blockade, indicating the presence of innate or acquired resistance to this therapeutic approach. We have been using xenografts of human rhabdomyosarcoma cell lines RD(embryonal) and RH30 (alveolar) to model both innate and acquired resistance to IGFIR blockade. Our xenograft models of rhabdomyosarcoma predicted short duration responses that mimicked the clinical observations, and we have been attempting to use these models to better understand how various signaling pathways in addition to IGFIR may interact and indeed be used as “by-pass” resistance pathways. Our hope is that this knowledge will lead to rational combination targeted therapy that we assume will be necessary for maximal clinical benefit. I will discuss one example of an additional signaling pathway that was activated upon IGFIR blockade, and how this information led to combination targeted therapy that holds therapeutic promise. We recently demonstrated that Src-Family Kinase (SFK) YES is highly expressed and functional in rhabdomyosarcomas. Since SFK signaling has been previously linked to IGF signaling in other tumor types, we evaluated the effect of IGFIR on YES activation. YES is rapidly activated upon IGFIR blockade using either a monoclonal antibody directed against the IGFIR or an IGFIR kinase inhibitor, suggesting that YES activation could play a role in resistance to IGFIR blockade. We next tested dual SFK and IGFIR inhibition and found that combined SFK and IGFIR inhibition led to more potent inhibition of both RD and RH30 rhabdomyosarcoma cell lines and perhaps most importantly led to increased apoptosis. Based on these promising in vitro effects, we evaluated the combination of IGFIR Ab, R1507 plus the SFK inhibitor, dasatinib, in our RD and RH30 xenografts. While both R1507 and dasatinib led to tumor growth inhibition as single agents, mice developed resistance to both single agents within 70-90 days of treatment. In contrast, the mice treated with the combination of R1507 and dasatinib did not develop resistance after 90 days and also showed enhanced tumor growth inhibition. This example highlights the need to better understand how signaling pathways interact so that one may predict consequences of therapeutic intervention using targeted agents and develop combinations that will undoubtedly be necessary to achieve the maximal clinical benefit of agents designed to inhibit specific signaling pathways. Citation Format: Lee J. Helman, Fernanda Arnaldez, Christine Heske, Xiaolin Wan, Choh Yeung. Combination targeted therapy in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA33.