Xianhui Rong

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

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 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

Abstract LB-237: Targeting HER3 and IGF1R in NRG1 high lung squamous cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HER3 is a member of the ErbB family of receptor tyrosine kinases. Aberrant activation of HER3 as a result of HER2 amplification or neuregulin 1 (NRG1) over-expression has been demonstrated to mediate constitutive activation of downstream oncogenic signals. LJM716 is a fully human IgG1 anti-HER3 monoclonal antibody. It locks HER3 in an inactive conformation and prevents HER3 dimerization with other ErbB family members. This unique mode of action enables LJM716 to block both ligand dependent and ligand independent HER3 activation. In search of new indications that may benefit from anti-HER3 therapies, we noted that squamous cell carcinomas (SCC) tend to have relatively higher NRG1 expression as compared to their adenocarcinoma counterparts. Highest NRG1 expression was observed in the lung SCC cell lines in cell line encyclopedia (CLE). A subset of the lung SCC cell lines with high NRG1 expression are moderately sensitive to LJM716, with HER3 activation detected in all of the sensitive lines. Surprisingly, phosphorylation of IGF1R rather than other HER family members tracked closely to HER3 phosphorylation in the lung SCC lines. Consistent with this finding, co-treatment with either IGF1R inhibitor, OSI-906, or anti-IGF1R antibody, Figitumumab, with LJM716 further enhanced LJM716 activity in the three NRG1-high lung SCC lines in vitro. The enhanced anti-tumor activity of LJM716 and Figitumumab is also observed in mouse xenografts generated from these lung SCC lines. Furthermore, pharmacodynamic analysis revealed increased inhibition of PI3K/mTOR signaling with the combined treatment of LJM716 and Figitumumab. These data suggest that NRG1- driven HER3 activation is a common feature in a subset of lung squamous cell carcinoma. Combination of anti-HER3 and anti-IGF1R is effective at suppressing PI3K/mTOR signaling and inhibit growth in these tumors. Citation Format: Qing Sheng, Maria Pinzon-Ortiz, Rita Das, Alan Huang, Xianhui Rong, Z. Alexander Cao. Targeting HER3 and IGF1R in NRG1 high lung squamous cell carcinoma. [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 LB-237. doi:10.1158/1538-7445.AM2014-LB-237