Duncan Walker

Mcl-1 Stability Determines Mitotic Cell Fate of Human Multiple Myeloma Tumor Cells Treated with the Kinesin Spindle Protein Inhibitor ARRY-520

Kinesin spindle protein (KSP/Eg5) inhibitors are novel anticancer agents that have thus far shown only modest activity in the clinic. Understanding how to identify patients who may be most sensitive to treatment is clearly needed to improve the development of these molecules. We studied four multiple myeloma cell lines treated with the KSP inhibitor ARRY-520 to identify factors important for initiating apoptosis while cells are arrested in mitosis. The majority (three of four) of cell lines underwent mitotic arrest, with apoptosis occurring in mitosis within 24 to 30 hours. The remaining line (NCI H929) is temporally refractory to ARRY-520 treatment, undergoing mitotic slippage and subsequently peaking in apoptotic markers after 72 hours of treatment, while most cells are in interphase. Interestingly, loss of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) coincided with mitotic cell death. Stabilization of Mcl-1 resulted in a delayed onset of apoptosis, whereas enforced downregulation of Mcl-1 increased cell death in response to KSP inhibition. Thus, variation in responses to KSP inhibition is governed by a balance between survival proteins and spindle checkpoint integrity. Cells relying on short-lived survival proteins during mitosis are more likely to undergo apoptosis in response to KSP inhibition. We propose that patients with hematologic malignancies, which rely on Mcl-1, would therefore be good candidates for treatment with KSP inhibitors. Mol Cancer Ther; 9(7); 2046–56. ©2010 AACR.

A diaminocyclohexyl analog of SNS-032 with improved permeability and bioavailability properties.

The identification of a selective CDK2, 7, 9 inhibitor 4 with improved permeability is described. Compound 4 exhibits comparable CDK selectivity profile to SNS-032, but shows improved permeability and higher bioavailability in mice.

A phase 1 dose‐escalation study of filanesib plus bortezomib and dexamethasone in patients with recurrent/refractory multiple myeloma

Filanesib is a kinesin spindle protein inhibitor that has demonstrated encouraging activity in patients with recurrent/refractory multiple myeloma. Preclinical synergy with bortezomib was the rationale for the current phase 1 study.

A Phase 1 and 2 study of Filanesib alone and in combination with low‐dose dexamethasone in relapsed/refractory multiple myeloma

Filanesib (ARRY‐520) is a highly selective inhibitor of kinesin spindle protein, which has demonstrated preclinical antimyeloma activity.

Modifications of the isonipecotic acid fragment of SNS-032: analogs with improved permeability and lower efflux ratio.

Modifications of the isonipecotic acid fragment of SNS-032 results in analogs which are more permeable and lower effluxed than SNS-032. The enantiomerically pure synthesis and the in vivo profile of analog 20 is described.

Tales of How Great Drugs Were Brought Down by a Flawed Rationale—Letter

In their recently published article ([1][1]), Komlodi-Pasztor and colleagues propose that development of selective mitotic inhibitors is grounded in a flawed therapeutic rationale. The authors argue that the primary determinant of sensitivity is tumor cell proliferation rate, and that these drugs

Abstract 1782: Human tumor explants are better predictors of clinical trial outcome than cell line xenografts for the KSP inhibitor ARRY-520

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Historically, human tumor cell line cultures and xenografts have served as standard models for identifying and optimizing anticancer drugs. Despite wide use, cell line models are poor predictors of clinical activity of drugs, particularly cytotoxics. Primary human tumor explant models have significant potential to inform clinical direction. These models have not undergone genetic drift or selection for growth in culture. Furthermore, low-passage explants retain the morphology and aspects of the tumor microenvironment reflecting the primary cancer in patients. These models further represent patients who have been treated with current chemotherapeutics. ARRY-520 is a novel kinesin spindle protein inhibitor which has been investigated in clinical studies in both solid and hematological cancers. In cell line xenografts, ARRY-520 has significant activity in solid tumors and even greater activity in hematological cell lines both in vitro and in vivo. In clinical studies, while ARRY-520 has demonstrated significant single-agent activity in multiple myeloma, activity in solid tumors has been modest. Thus, while preclinical data in hematological models has correlated with clinical activity in myeloma, the impressive solid tumor data has not translated to clinical activity in solid tumors. To probe the disparity of preclinical and clinical activity in solid tumors, we have investigated the ability of preclinical models to inform clinical success by retrospective analysis of the activity of ARRY-520 in solid tumor cell line and explant models, as compared to clinical data of this molecule in these indications. ARRY-520 shows potent activity in human tumor cell lines both in vitro, (IC50 0.5-14nM) and in vivo (50% tumor regression in 4/5 cell line xenografts CRC and PaCa including 100% regression in 2 models). By contrast, in 6 primary CRC and PaCa explants, ARRY-520 showed minimal activity (transient stable disease in 1/6 models and progressive disease in 5/6 models). Phase 1 solid tumor data on ARRY-520 was reported at ASCO 2010. In this study, 30 patients with solid tumors, treated at or above the MTD, were evaluable for response with best response of SD>12 weeks in 2 patients (7%). Within this study, there were 4 CRC and 3 PaCa patients, all of whom experienced progressive disease within 12 weeks of initiation. Further investigation of ARRY-520 in solid tumor explants is ongoing. Analysis of responsive and non-responsive models will be used to identify solid tumor types for further clinical development. In summary, in this study, human solid tumor explant xenografts more accurately predicted the actual clinical results from a Phase 1 solid tumor trial. These data suggest that solid tumor explant models may have significant utility in informing clinical decisions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1782. doi:1538-7445.AM2012-1782

Abstract 2550: Combination of the KSP inhibitor ARRY-520 with bortezomib causes sustained tumor regressions and significantly increased time to regrowth in bortezomib sensitive and resistant models of multiple myeloma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The allosteric kinesin spindle protein (KSP) inhibitor ARRY-520 has potent antitumor activity as a single agent in xenograft models of multiple myeloma, with complete response rates as high as 100%. It has shown promising signs of activity in a phase 1 trial in patients with advanced / refractory multiple myeloma, and a phase 2 study in this patient population is in progress. We report here on the striking activity of ARRY-520 in combination with bortezomib in multiple myeloma xenograft models. We investigated the activity of the combination of ARRY-520 with bortezomib in several xenograft models of multiple myeloma that are sensitive (Kas-6, ANBL-6, and H929) and resistant (RPMI8226, JJN3, a subline of ANBL-6 made resistant to bortezomib by in vitro treatment, and a subline of RPMI8226 that acquired a PI3K activating mutation) to bortezomib. The combination of ARRY-520 with bortezomib showed additive or superadditive activity, compared to either drug alone, in 6 of the 7 models tested. Complete responses and cures were observed in several models. The Kas-6 model was particularly sensitive, with a 100% cure rate (complete response through day 100). Since previous work from our lab (Mol. Cancer Ther. 9(7), 2046, 2010) implicates MCL1 status as a predictor of response to ARRY-520 in multiple myeloma cell lines, we evaluated the effect of the ARRY-520 + bortezomib combination on MCL1 and other markers of apoptosis. Treatment with the combination resulted in elevation of the cleaved / full length PARP ratio (up to 15X), compared to tumors treated with the single agents, along with a decline in MCL1 levels that coincided with elevation of the level of a 28kd fragment of MCL1 (up to 17X). This shortened form of MCL1 has been reported to be pro-apoptotic. Thus, the cleavage of MCL1 to a proapoptotic form, leading to a decrease in survival signaling and an increase in apoptotic signaling, may play a role in the activity of the ARRY-520 + bortezomib combination. The observation that the combination remained highly active even in models that were poorly responsive to single agent bortezomib suggests that the mechanisms underlying resistance to bortezomib may be distinct from those that contribute to activity of the combination. These results support ARRY-520 + bortezomib as a rational combination for clinical evaluation, including in patients with bortezomib-refractory disease. A phase 1 trial to evaluate the combination of ARRY-520 + bortezomib + dexamethasone is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2550. doi:10.1158/1538-7445.AM2011-2550

Abstract 3855: Investigation of the growth inhibitory activity of the MEK inhibitor ARRY-438162 in combination with everolimus in a variety of KRas and PI3K pathway mutant cancers

The RAS/RAF/MEK/ERK and PI3K signaling pathways are known to play key roles in cell growth, proliferation and survival. Targeted agents against factors in these pathways have been shown to provide therapeutic benefit in the treatment of cancer. However, there exists a considerable degree of cross-talk between these pathways such that when one pathway is inhibited the other is upregulated to sustain growth and survival signaling. Therefore, it would be expected that the concomitant use of targeted agents against both of these pathways would overcome the negative effects of this crosstalk, and enhance the ability of these agents to block cancer cell growth. We sought to understand whether or not the combination of the MEK inhibitor ARRY-438162 and the TORC1 inhibitor Everolimus would combine to enhance cell killing compared to the activity of the single agents. It has been established that in the majority of cancers, constituents of one or both of these pathways are mutated conferring constitutive activation to the pathway. Thus, we investigated whether key common mutations in either of these pathways conferred particular sensitivity or resistance to the combination. The growth inhibitory activities of ARRY-438162 and Everolimus were assessed alone and in combination in 26 different cell lines from a variety of human cancers with documented mutations in the KRAS, PTEN, PI3K and BRAF genes. In this regard, we report the identification of key genotypes that are associated with antagonistic, additive, and synergistic responses to the combination. In particular, treatment of the NCI-H460 NSCLC line [KRASG12D/PIK3CA] was only modestly responsive to either ARRY-162 or Everolimus as single agents (10% and 20% growth inhibition, respectively). However, the combination significantly inhibited the growth of this line, a result that was confirmed as synergistic by Bliss analysis. In order to validate the relevance of our in vitro data, an in vivo study was performed using NCI-H460 xenografts in nude mice. Everolimus alone showed no tumor growth inhibition while ARRY-438162 at doses of 30, 100 and 300 mg/kg, BID produced dose-related modest tumor growth inhibition (up to ∼50%). Combining the agents produced enhanced activity at all dose levels of ARRY-438162 without altering the exposure of either agent. Finally, in addition to growth inhibition, the biochemical effects of the single agent and the combination treatment on S6 protein, Erk, Akt, and various apoptosis regulator proteins were assessed as well as their effects on apoptosis and autophagy. ARRY-438162 having demonstrated significant tumor growth inhibitory activity in numerous pre-clinical models and acceptable preclinical and clinical safety profiles has advanced into Phase 1 clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3855.

Abstract 3610: Pan-ErbB inhibition by ARRY-334543 is superior to selective ErbB inhibition in a preclinical model that signals through multiple ErbB receptors

Activation of redundant receptor tyrosine kinases (RTKs) in tumors can confer resistance to selective RTK inhibitors. In particular, activation of multiple ErbB-family kinases is associated with diminished activity of selective EGFR or ErbB2 inhibitors. ErbB-family ligands are commonly expressed in cancers and can activate multiple redundant ErbB dimers. We hypothesize that ligand activation of multiple ErbB-family kinases in tumors can confer resistance to selective ErbB inhibitors, but not pan-ErbB inhibitors, suggesting a setting where pan-ErbB inhibitors may be advantageous over selective inhibitors. One such candidate currently in Phase 1/2 clinical trials is ARRY-334543, an oral, reversible pan-ErbB inhibitor with potent in vivo activity in both EGFR- and ErbB2-dependent human tumor xenografts. We tested this hypothesis in vitro with N87 gastric carcinoma cells that express high levels of constitutively active ErbB2 and low levels of EGFR. In the absence of EGF, ErbB2 homodimers are predominant and cells are highly sensitive to ErbB2 inhibition. ARRY-334543 and the ErbB2-selective inhibitor, ARRY-380, strongly inhibited N87 proliferation (IC50 = 60 nM and 5 nM, respectively) while the EGFR-selective inhibitor erlotinib was active only at high concentrations (IC50 = 860 nM). No benefit was observed by combining ARRY-380 and erlotinib. EGF-stimulation of N87 cells caused the formation of EGFR homodimers and EGFR/ErbB2 heterodimers which significantly alters cellular responses to selective ErbB inhibitors. The cellular IC50 for ARRY-380 increased 57-fold while erlotinib activity was relatively unchanged, suggesting a loss of dependence on ErbB2 signaling alone. The dual inhibition of EGFR and ErbB2, either by ARRY-334543 or the combination of ARRY-380 and erlotinib, largely maintained potency with only a modest increase in IC50 (9- to 10-fold). These data suggest that activation of EGFR can significantly diminish the activity of selective ErbB inhibitors, but not pan-ErbB inhibitors. Dual-responsiveness was also observed in vivo where both EGFR and ErbB2 homodimers were detected in N87 xenografts and dual EGFR/ErbB2 treatment resulted in superior tumor growth inhibition (TGI). ARRY-334543 was highly active with 90% TGI (100 mg/kg BID). Treatment with the combination of ARRY-380 and erlotinib (50 mg/kg QD each, MTD when given in combination) had similar activity with 82% TGI. Treatment with either erlotinib (100 mg/kg QD) or ARRY-380 (50 mg/kg QD) alone was less efficacious. These results suggest that the pan-ErbB inhibitor, ARRY-334543, can be differentiated from selective ErbB inhibitors by targeting tumors that signal through multiple ErbB-family receptors. A wide range of tumor types has been shown to activate multiple ErbB-family members and clinical evaluation of this hypothesis is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3610.