Maria Pinzon-Ortiz

RAD001 Enhances the Potency of BEZ235 to Inhibit mTOR Signaling and Tumor Growth

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.

Abstract S4-04: Overcoming resistance to PI3K inhibitors in PIK3CA mutant breast cancer using CDK4/6 inhibition: Results from a combinatorial drug screen

Various components of the phosphatidylinositol 3-kinase (PI3K) pathway are deregulated across a spectrum of human cancers. Notably, cancers with PIK3CA mutations, seen in roughly 30% of breast cancers, are amongst the most sensitive to PI3K inhibitors (PI3Ki) as single agents. Therefore, there have been great efforts to develop PI3K inhibitors specifically for these types of cancers, and many agents have already entered the clinic. Although initial responses and prolonged stable disease have been observed, resistance frequently emerges. Moreover, there is a subset of PIK3CA mutated cancers that unexpectedly do not exhibit an initial response or disease stabilization upon exposure to PI3K inhibitors, despite presence of the mutation. These cancers are said to have de novo resistance to PI3K inhibition. To determine methods of overcoming resistance to PI3K inhibitors, we generated two models with acquired resistance to the p110a isoform specific inhibitor BYL-719 (BYL) using MDA-MB-453 (453) and T47D. We also established one model of resistance to the pan-PI3K inhibitor GDC-0941 using MCF7 cells. These lines were chosen because of their PIK3CA mutated status and sensitivity to PI3K inhibition. Each cell line was grown in increasing concentrations of PI3K inhibitor until the cells proliferated readily at a dose of drug that effectively reduced cell viability and inhibited pAKT in the sensitive parental cell lines. Interestingly, both BYL resistant cells (453R and T47DR) were cross resistant GDC and the MCF7R line was refractory to BYL. To elucidate mechanisms to overcome resistance to PI3K inhibitors, we undertook a combinatorial drug screen, in which PI3K inhibitor resistant cells were treated with escalating doses of a panel of 45 targeted agents, both in the presence and absence of a fixed dose of PI3Ki, to determine which agents synergized effectively with PI3K inhibition in these resistant cells. We observed in each of the three PI3Ki resistant models a synergy between the CDK4/6 inhibitor LEE-011 and PI3K inhibition. We furthermore tested this combination of agents in a PIK3CA mutated breast cancer model with de novo resistance to PI3K inhibitors, CAL51, and again noted efficacy with the combination of GDC and LEE-011 while either agent on its own displayed minimal activity. To determine whether addition of CDK 4/6 inhibition might be an effective addition to PI3Ki in the upfront setting in vivo, we injected each of the PIK3CA mutated lines MCF7, 453, and T47D into female nude mice and treated with vehicle, BYL, LEE-011, or the combination. We noted in each of the three models that the combination of agents, led to tumor regression that was more substantial than single agent treatment, and furthermore delayed the acquisition of resistance relative to single agent therapy. We furthermore tested GDC with LEE-011 singly and in combination in both MCF7 and CAL51 xenografts and again noted that the combination of agents led to tumor regression, whereas in these instances, single agent treatment did not. We conclude that the combination of PI3K and CDK 4/6 inhibition may be an effective strategy for treating PIK3CA mutated breast cancer and deserves further study in the clinical setting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S4-04.

The identification and characterization of a STAT5 gene signature in hematologic malignancies

BACKGROUND The JAK-STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. Dysregulated JAK-STAT signaling has been implicated in the pathogenesis of multiple human malignancies. OBJECTIVE Given this pivotal role of JAK-STAT dysregulation, it is important to identify patients with an overactive JAK-STAT pathway for possible treatment with JAK inhibitors. METHODS We developed a gene signature assay to detect overactive JAK-STAT signaling. The cancer cell line encyclopedia and associated gene-expression data were used to correlate the activation status of STAT5 with the induction of a set of STAT5 target genes. RESULTS Four target genes were identified (PIM1, CISH, SOCS2, and ID1), the expression of which correlated significantly with pSTAT5 status in 40 hematologic tumor cell lines. In pSTAT5-positive models, the expression of the gene signature genes decreased following ruxolitinib treatment, which corresponded to pSTAT5 downmodulation. In pSTAT5-negative cell lines, neither pSTAT5 modulation nor a change in signature gene expression was observed following ruxolitinib treatment. CONCLUSIONS The gene signature can potentially be used to stratify or enrich for patient populations with activated JAK-STAT5 signaling that might benefit from treatments targeting JAK-STAT signaling. Furthermore, the 4-gene signature is a predictor of the pharmacodynamic effects of ruxolitinib.

Abstract 3684: The combination of JAK inhibitor, ruxolitinib, pan-PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical mouse model of myeloproliferative neoplasia

The JAK/STAT axis is a critical component downstream of multiple cytokine and growth factor receptor signaling pathways. The genetic aberration of JAK2V617F and the associated activation of STAT in myeloproliferative neoplasia (MPN) is one example of the involvement of this pathway in human cancer. Activated JAKs phosphorylate STAT proteins, which then up-regulate the transcription of STAT target genes such as PIM1. Pim kinases are involved in the regulation of cell cycle and proliferation. The inhibition of JAK1/2 by the JAK inhibitor, ruxolitinib (RUX), results in the suppression of the JAK-STAT pathway and promotes significant clinical benefit in patients with myelofibrosis. Our previous preclinical studies in a Ba/F3-JAK2V617F-driven MPN model demonstrate that the combination of RUX and the pan-PIM inhibitor, LGH447, exhibits greater inhibition of spleen weight and some reduction of JAK2V617F allele burden, compared with the RUX monotherapy. Mutant JAK2V617F has been shown to increase CDC25A transcription through activated STAT5. It also regulates p27 at both the gene expression and phosphorylation levels. The activation of CDC25A and the modulation of p27 have been postulated to trigger the activation of cyclin dependent kinases (CDK), such as CDK4, to initiate cell cycle progression. Additionally, activated STATs and Pims have been shown to activate D cyclins that are upstream of CDK4/6. Here, we explored the hypothesis that CDK4/6 inhibition, in conjunction with JAK and PIM inhibition, would enhance therapeutic efficacy against MPN. LEE011 is a potent and selective inhibitor of CDK4/6. The combination of RUX and LEE011 was tested in an MPN model with Ba/F3 cells harboring EPOR-JAK2V617F. While RUX monotherapy reduced spleen weight and total tumor burden by more than 50%, it had marginal effect on JAK2V617F allele burden in this model. The combination of RUX and LEE011 resulted in an additional 2 to 3 fold reduction in spleen weight and total tumor burden. Yet, no clear modulation of JAK2V617F allele burden was observed. To further enhance the anti-tumor activity, we tested the triple combination of RUX, LGH447 and LEE011 in this MPN model. This triple combination resulted in >99% reduction of total tumor burden and a ∼96% reduction of spleen weight. Furthermore, the triple combination of RUX, LGH447 and LEE011 significantly down-modulated JAK2V617F allele burden by > 80%. Our preclinical data indicate that the triple combination of RUX, CDK4/6 inhibitor, LEE011, and pan-PIM inhibitor, LGH447, may preferentially impact the JAK2V617F mutant MPN clones. This combination also achieves greater reductions in tumor burden and spleen weight in our preclinical MPN model. Therefore, potentially greater therapeutic benefit in subgroups of MPN patients may be achieved with the triple combination of RUX, LEE011 and LGH447. Citation Format: Maria Pinzon-Ortiz, Xianhui Rong, Abdel Saci, Robert Schlegel, Gary Vanasse, Giordano Caponigro, Z. Alexander Cao. The combination of JAK inhibitor, ruxolitinib, pan-PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical mouse model of myeloproliferative neoplasia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2014-3684

The potent and selective cyclin-dependent kinases 4 and 6 inhibitor ribociclib (LEE011) is a versatile combination partner in preclinical cancer models

Inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) is associated with robust antitumor activity. Ribociclib (LEE011) is an orally bioavailable CDK4/6 inhibitor that is approved for the treatment of hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer, in combination with an aromatase inhibitor, and is currently being evaluated in several additional trials. Here, we report the preclinical profile of ribociclib. When tested across a large panel of kinase active site binding assays, ribociclib and palbociclib were highly selective for CDK4, while abemaciclib showed affinity to several other kinases. Both ribociclib and abemaciclib showed slightly higher potency in CDK4-dependent cells than in CDK6-dependent cells, while palbociclib did not show such a difference. Profiling CDK4/6 inhibitors in large-scale cancer cell line screens in vitro confirmed that RB1 loss of function is a negative predictor of sensitivity. We also found that routinely used cellular viability assays measuring adenosine triphosphate levels as a proxy for cell numbers underestimated the effects of CDK4/6 inhibition, which contrasts with assays that assess cell number more directly. Robust antitumor efficacy and combination benefit was detected when ribociclib was added to encorafenib, nazartinib, or endocrine therapies in patient-derived xenografts.

Abstract 2343: The immune modulatory roles of IAP inhibitor, LCL161, and its connection to immune-checkpoint molecules

Tumor immunotherapy is a unique therapeutic modality in our fight against human cancers. The recent success of immune-checkpoint therapies highlights the value and potential of this approach. Previous studies have demonstrated positive immune-modulatory activities of the IAP inhibitor, NVP-LCL161. In this study, we sought to further explore the immune-modulatory activities of NVP-LCL161 in connection with immune-checkpoint molecules. In PBMC stimulation assays, NVP-LCL161 potently induced IFN and enhanced proliferation, while down-modulating IL-10 production. Additionally, the role of NVP-LCL161 on immune-checkpoint modulation was revealed in CyTOF analysis of in vitro stimulated PBMCs. NVP-LCL161 treatment led to increases in TIM-3 expression on multiple T cell subsets, and on cells of the myeloid lineage. These observations illustrate potential modulation of specific immune-checkpoint molecule by NVP-LCL161, thus suggesting possible synergy between TIM-3 immunecheckpoint blockade and NVP-LCL161. NVP-LCL161 was further combined in vivo with anti-PD1 antibody in the murine syngeneic tumor model, MC38. At multiple dosing schedules, synergy was observed with this combination, which appeared to be independent of single agent efficacy. Expression profiling of MC38 tumor tissues by a customized Nanostring panel revealed elevation of gene signatures in T cells, dendritic cells, cytokines and chemokines, amongst others. In summary, the preclinical data support the hypothesis that NVP-LCL161 is an active immune modulator. Its combination activity with immune_checkpoint inhibitors would warrant further exploration in both preclinical and clinical setting. Citation Format: Maria Pinzon-Ortiz, William Hastings, Tyler Longmire, Pamela Shaw, Xianhui Rong, Masato Murakami, Benjamin H. Lee, Glenn Dranoff, Kenzie MacIsaac, Z. Alexander Cao. The immune modulatory roles of IAP inhibitor, LCL161, and its connection to immune-checkpoint molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2343.

Abstract 2522: Molecular and cellular mechanisms underlying the therapeutic efficacy of the combination of JAK inhibitor, ruxolitinib, PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical model of myeloproliferative neoplasia

The JAK-STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. The genetic aberration of JAK2 V617F and the associated activation of STAT in myeloproliferative neoplasia (MPN) is one example of the involvement of this pathway in human cancer. We have previously shown the combination benefits of combining ruxolitinib (Jak1,2 inhibitor), with LGH447 (PIM inhibitor) and LEE011 (CDK4/6 inhibitor) in a Ba/F3-JAK2 V617F -driven MPN model. Here, we set to explore the molecular and cellular mechanisms underlying this combination benefit in the JAK2 V617F UKE-1 model of MPN. The triple combination of ruxolitinib, LGH447 and LEE011 resulted in sustained tumor regression in the UKE-1 model. Ruxolitinib, LGH447 and LEE011 inhibited pSTAT5, pBAD and pRb respectively. The monotherapies and the combination of ruxolitinib + LGH447 did not affect G1-S cycle. In contrast, the combination of ruxolitinib+LEE011, LGH447+LEE011 and the triple combination resulted in significant inhibition of G1-S progression. Consequently, after 72 hours of treatment, the percentage of cells in S phase decreased from 67% under DMSO to 15.8% under ruxolitinib+LEE011, and to 1.6% under the triple combination. Furthermore, monotherapies of LGH447 or LEE011 did not affect cell viability. Ruxolitinib monotherapy and ruxolitinib+LGH447 combination reduced viable cell% by 47% and 86%, respectively. The triple combination reduced it by 91%. Our data suggest that the triple combination efficacy is the result of both cell killing and cell cycle blockade. The inhibition of JAK-PIM pathway significantly reduced cell viability. And the inclusion of CDK4/6 inhibition by LEE011 lead to more potent cell killing and cell growth arrest. Taken together, the triple combination achieves sustained tumor regression in preclinical models of MPN. Citation Format: Maria Pinzon-Ortiz, Tyler Longmire, Xianhui Rong, Giordano Caponigro, Gary Vanasse, Benjamin H. Lee, Z. Alexander Cao. Molecular and cellular mechanisms underlying the therapeutic efficacy of the combination of JAK inhibitor, ruxolitinib, PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical model of myeloproliferative neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2522. doi:10.1158/1538-7445.AM2015-2522

Abstract 3489: Novel translational pharmacology approaches on dose reduction and alternative scheduling for the combination of JAK inhibitor, ruxolitinib, PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011 in a preclinical model of myeloproliferative neoplasia

The JAK-STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. It has been implicated in the pathogenesis of multiple human diseases. The genetic aberration of JAK2 V617F and the associated activation of STAT in myeloproliferative neoplasia (MPN) is one example of the involvement of this pathway in human cancer. We have shown the combination benefits of combining ruxolitinib (Jak1, 2 inhibitor), with LGH447 (PIM inhibitor) and LEE011 (CDK4/6 inhibitor) in a Ba/F3-JAK2 V617F -driven MPN model. This triple combination resulted in ∼99% reduction of total tumor burden and a ∼96% reduction of spleen weight. Furthermore, the triple combination of ruxolitinib, LGH447 and LEE011 reduced JAK2 V617F allele burden by > 80%. To translate this combination from preclinical setting to the clinic, it is critical to evaluate dose to efficacy relationship for each agents and scheduling to efficacy correlation for this combination. Here, the preclinical doses for ruxolitinib, LGH447 and LEE011 were determined, based on their clinically achieved exposure. We then examined “intermittent dosing” of this combination in the same preclinical model. Our data suggest that the combination efficacy of ruxolitinib-LGH447-LEE011 is dependent on continuous administration of the agents. Finally, we examined the effect of dose reduction for each of the three agents on the combination efficacy in the Ba/F3-JAK2 V617F -driven MPN model. By modifying the doses for ruxolitinib, LGH447 and LEE011 separately in the combination, our study reveals that the triple combination efficacy is most sensitive to LEE011 dose reduction, and it is least sensitive to LGH447 dose reduction. In summary, our studies aim to design novel preclinical approaches to inform and the design of clinical dose escalation in novel combination therapies. Citation Format: Maria Pinzon-Ortiz, Xianhui Rong, Gary Vanasse, Z. Alexander Cao. Novel translational pharmacology approaches on dose reduction and alternative scheduling for the combination of JAK inhibitor, ruxolitinib, PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011 in a preclinical model of myeloproliferative neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3489. doi:10.1158/1538-7445.AM2015-3489

Abstract 2692: High allele frequency of KRAS functional mutations predicts resistance to MEK inhibitors: Evidence from cell lines and human tumor xenograft models

Introduction KRAS mutant tumors represent a high unmet need for effective therapy. Even though there is no targeted therapy for KRAS, inhibitors of downstream targets like MEK have shown efficacy in preclinical studies. However, clinical trials of MEK inhibitors in KRAS-mutant tumors have not been successful, although there have been some responders. We sought to understand resistance to MEK inhibition in KRAS-mutant tumors and to develop biomarkers for patient stratification. Results Using DNA and RNA sequencing data from cell lines, we found that high variant allele frequency (>50%) of KRAS functional mutations (G12, G13, Q61) was associated with baseline resistance to MEK inhibitors AZD6244 and MEK162 across multiple tumor types. Low allele frequency of KRAS mutation was found to be necessary but not sufficient for sensitivity to MEK inhibitors, suggesting the existence of additional resistance mechanisms. Comparison of copy number and variant allele frequency of KRAS showed that MEK inhibitor-resistant cell lines have either amplified mutant alleles or lost WT alleles. This prediction of high KRAS mutant allele frequency being associated with MEKi resistance was successfully validated in vivo in 19 primary colorectal tumor xenograft models treated with MEK162. High KRAS mutant allele frequency and high KRAS expression were also associated with acquired resistance to BKM120+MEK162 combination treatment in an in vivo model of HeyA8, an ovarian cell line with a KRAS G12D mutation. This prediction was also supported by the observation in a Phase II clinical trial with MEK inhibitor refametinib + gemcitabine in pancreatic cancer [1]. Patients with disease progression had a significantly higher KRAS mutant allele frequency compared to partial responders and stable disease. Conclusion High allele frequency of KRAS functional mutations is associated with resistance to MEK inhibitors. Excluding patients with high KRAS mutant allele frequency has the potential to improve clinical trial outcomes and benefit patients, with the added benefit that it may not require any additional screening or novel biomarkers. References [1] Reiss et al., J Clin Oncol 32:5s, 2014 (suppl; abstr 4129) Citation Format: Anupama Reddy, Maria Pinzon-Ortiz, Adnan Derti, Joshua Korn, David Ruddy, Guizhi Yang, John Green, Hui Gao, Joseph Lehar, Giordano Caponigro, Z. Alexander Cao. High allele frequency of KRAS functional mutations predicts resistance to MEK inhibitors: Evidence from cell lines and human tumor xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2692. doi:10.1158/1538-7445.AM2015-2692

Abstract 782: Modulation of receptor tyrosine kinase signaling by cMET inhibitor, INC280, in cMETmut/amp gastric carcinoma cell line Hs746.T

Aberrant Receptor Tyrosine Kinase (RTK) activity is one of the hallmarks of cancer. The genetic alternations of ABL and EGFR are classical examples of RTK-dependent oncogenesis. Recent data suggest that non mutated RTK signaling can form feedback pathways to serve as resistant mechanism against targeted therapies. Here, we examined the regulation of RTK signaling by a potent cMET inhibitor, INC280 (enzymatic EC50 against c-MET = 0.13 nM), in a cMET-dependent human gastric tumor model Hs746.T. Hs746.T is a heavily cMET dependent cell line with both cMET amplication (12.9 copies) and mutation (G-T mutation at slice donor site to cause juxtamembrane domain deletion). This model is exquisitely sensitive to cMET inhibitor, INC280 in vitro (GI50 = 9 nM) and in vivo. The treatment of INC280 lead to significant inhibition of pcMET. Furthermore, the treatment of INC280 resulted in the suppression of pEGFR, though INC280 does not actively inhibit EGFR (enzymatic EC50 against EGFR > 10 microM). This result supports prior reports that cMET and EGFR can form physical complex, with cMET modulating EGFR activation. Consequently, INC280 inhibited downstream signaling nodes of pAKT, pS6RP, pMEK and pERK in Hs746.T. To examine the RTK signaling underlying possible treatment relapse, we treated Hs746.T cells chronically in vitro under increasing concentrations of INC280. After 8 weeks of treatment, Hs746.T-R cells were generated that grew at 600 nM of INC280. RTK signaling was examined in Hs746.T-R cells under treatment of INC280. As expected, cMET signaling was significantly curtailed in Hs746.T-R cells, as was pEGFR. Interestingly, pPDGFRb was elevated in Hs746.T-R, compared to the parental cell line. We hypothesize that the increased signaling through PDGFRb may serve as resistance mechanism to INC280 in Hs746.T-R cells. In summary, we have examined RTK signaling in the cMET-dependent Hs746.T cell line. Our finding supported the notion of RTK cross-talk between cMET and EGFR in this setting. Additionally, our data reveal that additional RTK signaling (PDGFRb) can be induced under chronic treatment of INC280 to possibly mediate resistance to cMET inhibition. Citation Format: Maria C. Pinzon-Ortiz, Xianhui Rong, Jinsheng Liang, Hui Qin Wang, Alan Huang, Robert Schlegel, Z. Alexander Cao. Modulation of receptor tyrosine kinase signaling by cMET inhibitor, INC280, in cMETmut/amp gastric carcinoma cell line Hs746.T. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2015-782