Nikoo Falahatpisheh

Species-Specific Metabolism of SGX523 by Aldehyde Oxidase and the Toxicological Implications

An investigation was conducted to follow up on the apparent species-dependent toxicity reported for 6-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylthio)quinoline (SGX523), a mesenchymal-epithelial transition factor (c-MET) inhibitor that entered clinical development for the treatment of solid tumors. Patients treated with SGX523 exhibited compromised renal function presumably resulting from crystal deposits in renal tubules. Our independent metabo‘lite profiling of SGX523 indicates that a major NADPH-independent, late-eluting metabolite [6-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylthio)quinolin-2(1H)-one (M11)] was generated by monkey and human liver S-9, and to a lesser extent by rat S-9, whereas M11 was absent in dog S-9 incubations. We confirmed the identity of M11 as 2-quinolinone-SGX523. Experiments with various molybdenum hydroxylase inhibitors showed that aldehyde oxidase (AO), and not xanthine oxidase, metabolized SGX523 to M11 in monkey and human liver cytosol. In addition, the oxygen incorporated into M11 was derived from water rather than atmospheric oxygen, corroborating M11 formation via AO. After oral dosing in monkeys, metabolite profiling of plasma and urine showed that SGX523 was indeed metabolized to M11 and its N-demethyl analog (M8). In urine, M11 levels were ∼70-fold greater than that of SGX523, and the solubility of M11 in urine was only 3% of that of SGX523. In summary, SGX523 is metabolized by AO in a species-specific manner to a markedly less-soluble metabolite, M11. We propose that M11 is likely involved in the observed obstructive nephropathy reported in clinical studies. Moreover, this study illustrates the need to conduct thorough metabolic evaluations early in drug development to select the most relevant nonclinical species for toxicological evaluation.

Abstract 3523: Discovery of a novel BET inhibitor INCB054329

Bromodomains (BD) are protein modules that bind acetylated lysine residues and are components of many epigenetic modifiers and transcription factors. The BET (Bromodomain and extra-terminal) family is composed of four members each harboring two tandem BDs. BET proteins are critical regulators of transcription through interactions with complexes including Mediator and p-TEFb at gene promoter and enhancer elements. Studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins is therapeutic in models of cancer and acute inflammation. We describe the preclinical activity of a novel BET inhibitor INCB054329 for the potential treatment of malignant diseases. INCB054329 inhibited binding of BRD2, BRD3 and BRD4 to an acetylated histone H4 peptide with low nanomolar potency. In myeloma cell lines, treatment with INCB054329 inhibited expression of c-MYC and induced HEXIM1. The majority of myeloma, AML, and lymphoma cell lines tested were growth inhibited by INCB054329 with potencies less than 200 nM. Selectivity was seen when compared with nontransformed cells as the potency for growth inhibition of IL-2 stimulated T-cells from normal donors was greater than 1300 nM. Cell cycle analysis revealed treatment-induced G1 arrest. Furthermore in both AML and lymphoma cell lines, INCB054329 induced apoptosis consistent with increased expression of pro-apoptotic regulators. In vivo, oral administration of INCB054329 inhibited tumor growth in several models of hematologic cancers. In the MM1.S multiple myeloma xenograft model, inhibition of tumor growth was correlated with reduction of c-MYC levels. PK-PD analysis showed c-MYC suppression was associated with an IC50 value of less than 100 nM in vivo. In summary these studies demonstrate that INCB054329 is a potent inhibitor of BET transcriptional regulators in models of hematologic malignancies in vitro and in vivo and support its clinical development for the treatment of cancer. Citation Format: Phillip CC Liu, Xuesong Mike Liu, Matthew C. Stubbs, Thomas Maduskuie, Richard Sparks, Nina Zolotarjova, Jun Li, Xiaoming Wen, Margaret Favata, Patricia Feldman, Alla Volgina, Darlise DiMatteo, Robert Collins, Nikoo Falahatpisheh, Padmaja Polam, Yu Li, Maryanne Covington, Sharon Diamond-Fosbenner, Richard Wynn, Timothy Burn, Kris Vaddi, Swamy Yeleswaram, Andrew P. Combs, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Discovery of a novel BET inhibitor INCB054329. [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 3523. doi:10.1158/1538-7445.AM2015-3523

Abstract 692: The BET inhibitor INCB054329 is synergistic with JAK1 inhibition in models of multiple myeloma

Bromodomain and Extra Terminal (BET) protein inhibitors have emerged as a potentially effective therapeutic option for multiple tumor types, through their ability to regulate expression of genes necessary for proliferation and survival. For example, multiple myeloma (MM) cells have been shown to be highly sensitive to BET inhibition due in large part to the ability of BET proteins to control transcription of c-myc, an oncogene known to be dysregulated in MM. Likewise, some inflammatory response and cytokine signaling pathways associated with MM (eg. IL-6/JAK/STAT pathway) have also been shown to be reliant on BET proteins. Therefore, inhibition of both BET proteins and the JAK/STAT signaling pathway may be beneficial to MM patients. Here we assess the in vitro and in vivo effects of combining clinical compounds that target BET proteins and JAK in multiple myeloma cell lines. Studies were performed using the potent pan-BET inhibitor INCB054329 and selective JAK1 inhibitors. When tested in cell proliferation assays, the combination of BET and JAK1 inhibitors displayed strong synergistic effects in the IL-6 dependent INA-6 MM cell line in vitro. Western blots also revealed that several pharmacodynamic (PD) markers including c-MYC, PIM-2 and phospho-STAT3 were further repressed with the combination than with single agents alone. Likewise, the c-MYC and p-STAT3 PD markers could also be increasingly repressed in vivo by combined administration of BET and JAK1 inhibitors in the INA-6 mouse xenograft model. In vivo efficacy experiments in the INA-6 model resulted in enhanced, synergistic tumor growth inhibition in the BET/JAK inhibitor cohort as compared with the single drug cohorts. Interestingly, the cytokine independent MM1.S cell line was also sensitive to the BET/JAK inhibitor combination in vivo, while being far less sensitive to JAK1 inhibition as a monotherapy. In the MM1.S model, the c-MYC and p-STAT3 PD markers also behaved as seen in the INA-6 model. Our data indicate that the pharmacological inhibition of BET proteins and JAK1 yields strong combinatorial effects in MM cell lines both in vitro and in vivo. Therefore, dual inhibition of BET proteins and the JAK/STAT signaling pathway may offer a novel therapeutic approach and suggest a potential clinical utility for this drug combination in MM. Citation Format: Matthew C. Stubbs, Xuesong M. Liu, Xiaoming Wen, Jun Li, Valerie Dostalik, Sybil O9Connor, Eian Caulder, Margaret Favata, Mark Rupar, Yu Li, Beth Rumberger, Thomas Maduskuie, Richard Sparks, Nikoo Falahatpisheh, Padmaja Polam, Kris Vaddi, Timothy Burn, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. The BET inhibitor INCB054329 is synergistic with JAK1 inhibition in models of multiple myeloma. [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 692. doi:10.1158/1538-7445.AM2015-692

Abstract 5071: Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies

Inhibitors of the Bromodomain and Extra-Terminal (BET) family of bromodomain containing proteins regulate expression of key cell fate, cell cycle, and survival genes including c-myc. In preclinical models, BET inhibitors have demonstrated significant efficacy in a variety of different oncology indications, including hematological malignancies. Here we describe the preclinical profile of the novel, orally bioavailable BET inhibitor INCB057643 in preclinical models of hematologic malignancies. INCB057643 inhibited binding of BRD2/BRD3/BRD4 to an acetylated histone H4 peptide in the low nM range, and was selective against other bromodomain containing proteins. In vitro analyses showed that INCB057643 inhibited proliferation of human AML, DLBCL, and multiple myeloma cell lines, with a corresponding decrease in MYC protein levels. Cell cycle analyses indicated that G1 arrest and a concentration-dependent increase in apoptosis were seen within 48 hours of treatment with INCB057643. BRD proteins also regulate the expression of many pro-inflammatory genes. Production of several cytokines, including IL-6, IL-10 and MIP-1α, was repressed by INCB057643 in human and mouse whole blood stimulated ex vivo with LPS. Consistent with these effects, analyses of gene expression in cells treated with INCB057643 revealed that pathways involved in cell cycle progression, apoptosis, and IL-6 were among the most significantly altered in vitro. Oral administration of INCB057643 resulted in significant anti-tumor efficacy in xenograft models of AML, myeloma, and DLBCL. Additionally, combining INCB057643 with standard of care agents used for the treatment of DLBCL including rituximab and bendamustine resulted in enhanced anti-tumor efficacy relative to that achieved with single agent therapies at doses that were well tolerated. In addition, many B cell malignancies are reliant on the PI3Kδ pathway for proliferation and survival, suggesting that the combination of INCB057643 with the clinical stage PI3Kδ specific inhibitor INCB050465 may be a rational therapeutic strategy for DLBCL. Compared with single agent BETi or PI3Kδi therapy, the combination significantly potentiated tumor growth inhibition in DLBCL models representative of the ABC subtype (HBL-1), and the double hit GCB subtype (WILL2). These data suggest that clinical exploration of INCB057643 as a monotherapy or in combination in hematologic malignancies is warranted. Citation Format: Matthew C. Stubbs, Thomas Maduskuie, Timothy Burn, Sharon Diamond-Fosbenner, Nikoo Falahatpisheh, Alla Volgina, Nina Zolotarjova, Xiaoming Wen, Patricia Feldman, Mark Rupar, Robert Collins, Cindy Marando, Bruce Ruggeri, Maryanne Covington, Xuesong Mike Liu, Richard Wynn, Swamy Yeleswaram, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Andrew P. Combs, Phillip C. Liu. Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2017-5071

Abstract C103: The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models

Understanding the in vivo responses to immunoregulatory agents provides a basis for building more efficacious combination regimens. Pharmacologic inhibition of the oncogenic PI3Kδ pathway has been shown to be active in patients with hematopoietic malignancies. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, through the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses, suggesting that it may have additional utility in this patient population. We identified a similar immunomodulatory role for the PI3Kδ-selective inhibitor INCB050465 in a preclinical model of pancreatic cancer, where an increase in the number of CD8+ T cells, a decrease in the number of suppressor cells and efficacy were seen. Therefore we explored the potential of INCB050465 in additional preclinical solid tumor models, alone and in combination with other immunotherapeutic agents. INCB050465 inhibited tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition was not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate immune-mediated mechanisms, tumors were analyzed for modulation of gene expression and immune phenotype after mice received short-term treatment. INCB050465 was shown to significantly downregulate the T cell gene signature in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. As seen previously, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T-cell-inflamed model by reducing both Treg and M2 macrophages, which promotes re-activation of both CD4+ and CD8+ T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell response over time. In summary, pharmacological inhibition of PI3Kδ can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and complex cellular responses may provide rationale to identify individuals who may benefit from PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Liang-Chuan Wang, Michael Hansbury, Yue Zhang, Gengjie Yang, Timothy Burn, Paul Waeltz, Mark Rupar, Eddy Yue, Brent Douty, Thomas Maduskuie, Nikoo Falahatpisheh, Yun-long Li, Andrew Combs, Gregory Hollis, Reid Huber, Peggy Scherle. The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C103.

Pharmacological inactivation of PI3Kδ in the tumor microenvironment enhances efficacy of other immunotherapeutic agents

Pharmacological inhibition of the oncogenic PI3Kδ pathway has been shown to be efficacious in patients with hematopoietic malignancies. However, its therapeutic application in patients with solid tumors has not yet been tested. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, resulting from the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses. Therefore we explored the immunotherapeutic potential of our PI3Kδ-selective compound, INCB050465, in multiple preclinical tumor models. We demonstrate that INCB050465 can block tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition is not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate the immune-mediated mechanisms, tumors exposed to vehicle or INCB050465 were harvested and analyzed for modulation of gene expression and immune phenotype. INCB050465 was shown to significantly downregulate T cell gene signatures in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. In contrast, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. We next examined INCB050465 in combination with other immune modulators. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T cell-inflamed model by reducing both Treg and M2 macrophages, which then allowed the re-activation of both CD4 and CD8 T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell responses over time. In summary, inactivation of PI3Kδ with a pharmacological inhibitor can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and associated cellular responses may provide a rationale to identify individuals who may benefit most from PI3Kδ inhibitor-based immunotherapy combinations in the clinic.

Abstract 3525: The BET inhibitor INCB054329 is efficacious as a single agent or in combination with targeted agents in colorectal cancer models

The Bromodomain and extra-terminal (BET) family of proteins consists of BRD2, BRD3, BRD4 and BRDT. Each BET protein contains two bromodomains (BD1 and BD2) that recognize acetylated histones at enhancer and promoter regions of a select number of genes. BET proteins function as transcriptional regulators that are critical for the expression of oncogenes such as c-Myc; thus, BET proteins are important for cancer cell proliferation and survival. We have identified INCB054329, a potent and selective BET protein inhibitor that targets BD1 and BD2 of BRD2, BRD3 and BRD4. In this study, we characterized the pharmacology of INCB054329 in solid tumor cell lines. In a panel of colon cancer cell lines, more than 50% are sensitive to INCB054329 treatment with IC50 values below 500 nM in cell proliferation assays. INCB054329 down-regulated c-Myc expression, and induced cell cycle arrest and apoptosis in sensitive colon cancer cell lines. Moreover, INCB54329 was efficacious in the RKO colon cancer xenograft model. To understand whether BRD inhibition would synergize with other signaling pathway inhibitors and standard of care agents for colon cancer, we employed a high throughput combination screening strategy. Several combinations were active in a panel of colon cancer cell lines and demonstrated synergistic interactions based on combination index values. As an example, strong synergy was observed between INCB054329 and MEK inhibitors. The combination of INCB054329 and MEK inhibitors synergistically blocked expression of c-Myc protein and inhibited the MEK/ERK signaling pathway. Our data suggest the potential utilization of INCB054329 as a single agent or in combination with other targeted therapies for the treatment of colon cancer. Citation Format: Xuesong Liu, Jun Li, Xin He, Matthew Stubbs, Margaret Favata, Xiaoming Wen, Hong Chang, Beth R. Rumberger, Yanlong Li, Thomas Maduskuie, Richard Sparks, Nikoo Falahatpisheh, Padmaja Polam, Andrew P. Combs, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip C. Liu. The BET inhibitor INCB054329 is efficacious as a single agent or in combination with targeted agents in colorectal cancer 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 3525. doi:10.1158/1538-7445.AM2015-3525

Abstract 691: Activity of the BET inhibitor INCB054329 in models of multiple myeloma

Multiple myeloma (MM) is a disease of plasma cell transformation. Current therapy for MM is initially effective, but nearly all tumors relapse, making new therapeutic options a necessity. Due to their efficacy in MM models, small molecule inhibitors of Bromodomain and Extra Terminal (BET) proteins have generated much interest as potential therapeutic agents for MM. Efficacy from BET inhibitors in MM is thought to be driven by their ability to reduce transcript levels of the c-myc oncogene. Current MM standard of care (SoC) therapeutics include lenalidomide (Revlimid, Celgene), bortezomib (Velcade, Takeda), and Melphalan (Alkeran, GlaxoSmithKline). In preparation for potential clinical studies, these MM SoC agents were each combined with a potent pan-BET inhibitor, INCB054329, for in vitro and in vivo studies. In vitro, treatment of MM cell lines with INCB054329 inhibited expression of c-MYC, induced HEXIM1 levels and inhibited cell growth with potencies less than 200 nM. Combination of INCB054329 with SoC therapeutics showed synergistic effects in blocking MM cell proliferation. INA-6 and MM1.S mouse xenograft models of MM were utilized to study the effects of the combination of INCB054329 with the MM SoC agents in vivo. In each instance (INCB054329/lenalidomide, INCB054329/bortezomib, INCB054329/melphalan), additive to synergistic effects, as measured by inhibition of subcutaneous tumor growth, were seen in both models of MM. Since lenalidomide has recently been shown to bind the ubiquitin ligase cereblon (CRBN), leading to the degradation of two Ikaros family members, IKZF1 and IKZF3, that are essential to B cell fate and survival, we examined whether there could be functional overlap between BET inhibition and the downstream effects of lenalidomide. From MM1.S xenograft pharmacodynamic assays, we found a synergistic repression of c-MYC protein levels at 3hr post dose for the combination of INCB054329 and lenalidomide. Interestingly, the sharp decline in c-MYC protein levels in the MM1.S model appears to be independent of the IRF4 pathway, as IRF4 protein levels do not decrease until 24 hrs post dose of lenalidomide. Overall, our data indicate that INCB054329 may provide a novel combination partner with current standard of care therapies for MM, and support the clinical evaluation of the compound within the anti-myeloma treatment landscape. Citation Format: Matthew Stubbs, Xiaoming Wen, Valerie Dostalik, Sybil O9Connor, Eian Caulder, Alla Vogina, Thomas Maduskuie, Richard Sparks, Taisheng Huang, Nikoo Falahatpisheh, Padmaja Polam, Chu-Biao Xue, Xuesong M. Liu, Timothy Burn, Kris Vaddi, Andrew P. Combs, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. Activity of the BET inhibitor INCB054329 in models of multiple myeloma. [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 691. doi:10.1158/1538-7445.AM2015-691