Allison Drew

The Discovery and Optimization of a Novel Class of Potent, Selective, and Orally Bioavailable Anaplastic Lymphoma Kinase (ALK) Inhibitors with Potential Utility for the Treatment of Cancer.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

Comparison of 2 Cell-Based Phosphoprotein Assays to Support Screening and Development of an ALK Inhibitor

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY1604 ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen®) and phosflow platforms were used to detect modulation of pY1604 ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY1604 ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen® assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen® and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY1604 ALK.

EZH2 Inhibition by Tazemetostat Results in Altered Dependency on B-cell Activation Signaling in DLBCL

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

Abstract 1795: Characterization of a novel series of potent, selective inhibitors of wild type and mutant/fusion anaplastic lymphoma kinase

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that has been implicated as a driving oncogene in a number of cancers, including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma and inflammatory myofibroblastic tumors (IMT). Numerous genetic aberrations at the ALK locus are observed in cancer including point mutations, amplifications, translocations and inversions. Inversions are exemplified by inv(2)(p21;p23), which leads to the constitutively active oncogenic fusion protein EML4-ALK present in ∼5% of NSCLC. Crizotinib, a dual cMet/ALK kinase inhibitor, was recently approved by the FDA for locally advanced or metastatic NSCLC that is ALK-positive, thereby validating ALK as therapeutic target. Here we describe the pharmacological characterization of a novel series of potent, selective and orally bioavailable ALK kinase inhibitors. Members of this series inhibit wild type ALK, NPM-ALK fusion and crizotinib resistant ALK[L1196M] kinase activity at sub-nanomolar concentrations, displaying up to ∼200 fold increased inhibitory activity over crizotinib. Kinase profiling indicate that members of this series display increased selectivity scores relative crizotinib. In Karpas-299 cells, selected compounds inhibited both pY1604 ALK activation (IC50 = 2 nM) and cell proliferation (IC50 = 1 nM). Members of this series were also evaluated in the EML4-ALK expressing NSCLC cell line H3122, and displayed equipotent inhibition of pY1604 ALK activation and inhibition of cell proliferation (both IC50 = 1 nM). Members of this class did not inhibit growth of an ALK negative lymphoma cell line (HT). The in vivo activity of this series was examined in the Karpas-299 ALCL xenograft model. Compound was dosed daily (PO) at 10, 30 and 60 mg/kg. Tumor growth inhibition was observed at all dose levels, and the highest dose level resulted in significant tumor regression (96%, p 80% pALK inhibition was observed at the lowest doses tested, and complete inhibition was seen at doses of 30 mg/kg and above. In a direct comparison, members of this series achieved ALK inhibition in these tumors at a ∼15 fold lower plasma concentration than crizotinib. A PK/PD time course study was performed in the Karpas-299 model. A single 60 mg/kg dose of an inhibitor was able to maintain >90% ALK inhibition in tumors up to 24 hours post-dose, indicating significant tumor penetration and sustained ALK kinase inhibition. In conclusion, the described compounds are potent and selective inhibitors of ALK kinase, possess an impressive efficacy profile and drug-like pharmacokinetic properties. These features together indicate the potential for significant advantages over crizotinib. There is a compelling case for their clinical evaluation in patients with ALK-driven cancers. 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 1795. doi:1538-7445.AM2012-1795

Abstract 5060: Activity of the EZH2 inhibitor tazemetostat as a monotherapy and in combination with multiple myeloma therapies in preclinical models

The EZH2 inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase 2 clinical trials for the treatment of non-Hodgkin’s Lymphoma (NHL). EZH2 inhibitors have shown anti-proliferative effects in multiple preclinical models of NHL and objective clinical responses have been reported in patients with B-cell lymphomas in phase 1 and phase 2 studies of tazemetostat. Mounting evidence suggests that EZH2 is an important regulator of B cell differentiation, both in normal B-cells and in B-cell lymphoma, and may be an important mediator of cell fate in B-cell malignancies in the clinic. Consistent with its essential role in regulating B cell differentiation, recent studies have also shown a dependence on EZH2 activity in multiple myeloma (MM), a disease arising from terminally-differentiated B-cell lymphocyte plasmablasts. Frequent genetic alterations of epigenetic modulators are observed in MM, pointing towards an important role in the initiation and maintenance of this disease. Dysregulation of the H3K27 methyltransferase EZH2, its corresponding histone demethylase UTX and the H3K36 methyltransferase WHSC1 in MM suggest that disruption of the balance of histone methylation may be fundamental to MM pathogenesis in a subset of cases. Indeed, inhibition of EZH2 alone has shown potent anti-proliferative effects both in in vitro and in vivo preclinical models of MM. Here, we describe the effects of small molecule EZH2 inhibitors as monotherapy and in combination with standard of care agents in preclinical models of MM. Tazemetostat selectively inhibits intracellular H3K27 methylation in MM cell lines and elicits a robust anti-proliferative effect in 14-day assays. Following demonstration of single agent activity, we then investigated potential for combinatorial activity of tazemetostat with first and second line therapies for multiple myeloma as well as other non-approved but emerging therapies. Synergistic anti-proliferative activity was observed when tazemetostat was combined with glucocorticoid receptor agonists (dexamethasone, prednisolone), small molecule immune system modulators (lenalidomide, pomalidomide) and proteasome inhibitors (bortezomib, ixazomib) when cells were primed with tazemetostat for seven days prior to the addition of the standard of care drugs. Combination activity was also observed with an alternate treatment schedule where cells were co-treated with tazemetostat along with the combination partner for seven days. Studies with selected therapeutic modalities were expanded into in vivo xenograft models to further evaluate monotherapy and combination activity of EZH2 inhibitors in MM. Citation Format: Allison E. Drew, Vinny Motwani, John E. Campbell, Cuyue Tang, Jesse J. Smith, Richard Chesworth, Robert A. Copeland, Alejandra Raimondi, Scott Ribich. Activity of the EZH2 inhibitor tazemetostat as a monotherapy and in combination with multiple myeloma therapies in preclinical models [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 5060. doi:10.1158/1538-7445.AM2017-5060

Abstract C87: EZH2 inhibition leads to decreased proliferation in SMARCA4-deleted ovarian cancer cell lines

Introduction: The H3K27 histone methyltransferase EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), and is amplified, overexpressed, or mutated in multiple cancer types, supporting its function as an oncogene. In addition to genetic alterations in EZH2 itself, distal genetic changes in other proteins can lead to oncogenic dependency on EZH2 activity. For example, we have previously established that cell lines and xenografts deficient in INI1 (SNF5/SMARCB1), a core component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, display profound sensitivity and durable regressions in the presence of the selective EZH2 inhibitor tazemetostat (EPZ-6438). Intriguingly, a complete response was observed in a patient with an INI1-negative rhabdoid tumor who participated in the tazemetostat Ph1 dose escalation study. This suggests that these tumors are addicted to dysregulated PRC2 activity, and confirms the previously proposed antagonistic relationship of SWI/SNF with PRC2, which is perturbed in INI1-deficient tumors. The loss of INI1 induces inappropriate SWI/SNF function, abrogating the repression of PRC2 activity, resulting in Polycomb target genes, such as those involved in differentiation and tumor suppression, to become aberrantly repressed. In addition to deletion of INI1, there are numerous reports describing genetic alterations in other SWI/SNF complex members. Given the oncogenic dependency of INI1-deficient tumors on PRC2 activity, we sought to investigate the sensitivity of other SWI/SNF mutated cancer types to EZH2 inhibition. Specifically, we investigated the effects of EZH2 inhibition in ovarian cancers carrying somatic mutations in the SWI/SNF complex members ARID1A and SMARCA4. Methods and results: A panel of ovarian cancer cell lines of different histologies was subjected to proliferation assays in 2-D tissue culture for 14 days in the presence of increasing concentrations of an EZH2 inhibitor. Selected cell lines were also tested in 3-D cultures, as it has been suggested in the literature that this context is necessary to observe anti-proliferative effects with EZH2 inhibitors. We found that ovarian cancer cell lines deficient in the SWI/SNF component SMARCA4 (also known as BRG1) are among the most sensitive in response to EZH2 inhibition, as demonstrated by decreased proliferation and/or morphology changes, at concentrations that are clinically achievable. In contrast, mutations in ARID1A, another SWI/SNF component, do not broadly confer sensitivity to EZH2 inhibition in ovarian cancer cell lines in either 2-D or 3-D in vitro assays. Furthermore, the effects of EZH2 inhibition on SMARCA4-negative ovarian cancer cells are context specific, since other cell types with SMARCA4 deletion, such as lung carcinoma cell lines, do not exhibit anti-proliferative affects with EZH2 inhibitor treatment. Conclusions: These data suggest that tazemetostat may have therapeutic benefit in SMARCA4-deleted ovarian cancer, such as small cell cancer of the ovary of the hypercalcemic type (SCCOHT), which shows a high degree of loss of SMARCA4 expression. Further in vitro and in vivo studies are underway to interrogate these initial results further. Citation Format: Sarah K. Knutson, Allison E. Drew, Christopher Plescia, Robert A. Copeland, Jesse J. Smith, Heike Keilhack, Scott Ribich. EZH2 inhibition leads to decreased proliferation in SMARCA4-deleted ovarian cancer cell lines. [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 C87.

Abstract 406: CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships

Target identification is a critical step in drug discovery, but the process has many challenges including non-specific reagents, limited ability to test numerous models, and incomplete target inhibition. Pooled screening with CRISPR/Cas9 permits the quick and accurate examination of proliferation effects across many genes and many cell lines. To determine the specific dependencies of cell lines on epigenetic pathways, we designed a CRISPR/Cas9 library to target 640 epigenetic genes and screened more than 200 cell lines covering a variety of oncology indications, including breast, lung, and renal cell carcinoma (RCC). We find that CRISPR pooled screening is a highly effective approach for target identification and provides robust, highly reproducible data as long as a sufficient number of small guide RNAs are used. We identify known pan-essential genes, including in the transcription (CDK9), translation (EIF4A1 and EIF4A3) and splicing (SRSF2) machinery. We additionally identify many novel pan-essential genes across a variety of epigenetic pathways, including histone acetylases and deacetylases, chromatin remodeling factors, helicases and others. We also investigated epigenetic synthetic lethal interactions that have been previously reported. For example, it has been reported that the SWI/SNF family displays paralog synthetic lethality for SMARCA2 in the context of SMARCA4 mutations, and for ARID1B in the context of ARID1A mutations. While we do see that some of the same trends hold, the synthetic lethal relationship appears to be more complex than previously realized, including the need to examine mRNA levels in addition to mutation type. Most importantly, we identify more than 100 epigenetic genes which show selective sensitivity, i.e. where knockout shows an anti-proliferative effect in only a subset of the cell lines. These are the most promising targets for further drug discovery programs. We have used additionally CRISPR/Cas9-domain based screening to identify the functionally relevant sites for many of these genes. Furthermore, we can overlay gene expression and mutation data to identify novel synthetic lethal relationships. One gene that displays selective sensitivity is EGLN1, the prolyl hydroxylase for the hypoxia-inducible factor, HIF1α. We find that EGLN1 is required for proliferation only in RCC cell lines which retain wild-type VHL, another component of the hypoxia response pathway, which is frequently lost in RCC. As such, EGLN1 loss is synthetically lethal in the presence of wild-type VHL in RCC cells. Thus this approach not only identifies an enzymatic drug target but also a patient stratification method. Other novel synthetic lethal interactions have also been identified. Our data demonstrates that CRISPR pooled screening is a powerful technique for identification of epigenetic synthetic lethal interactions. Citation Format: Alexandra R. Grassian, Darren Harvey, Julian Fowler, Allison E. Drew, Igor Feldman, Richard Chesworth, Robert Copeland, Jesse J. Smith, Scott Ribich. CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships [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 406. doi:10.1158/1538-7445.AM2017-406

Abstract 5376: Identification of the first small molecule PRMT6 inhibitor tool compound

PRMT6 is a nuclear-localized arginine methyl transferase (RMT) capable of adding omega-N(G)-monomethylarginine and asymmetric omega-N(G),N(G)-dimethylarginine derivatives to histone and other protein substrates containing a GAR motif. PRMT6 can methylate both histone and non-histone substrates and is the only RMT known to methylate the H3R2 mark. This mark can act in opposition to the activating H3K4me3 mark, effectively acting as a transcriptional repressor. PRMT6 overexpression has been reported in several cancer types including melanoma and bladder, lung, and prostate carcinoma suggesting that inhibition of PRMT6 activity might have therapeutic utility. However, until now no small molecule PRMT6 inhibitor has been available to carry out in vitro and in vivo studies. Here we report identification of an aryl pyrazole as the first small molecule PRMT6 inhibitor. This tool compound has an IC50 of 10nM in a PRMT6 biochemical assay and the binding mode has been elucidated through crystallography. The compound inhibited methylation of H3R2 in a dose dependent manner in an engineered model in which PRMT6 was transiently expressed in the A375 cell line. The tool compound showed a half-life (t½) of 8.54 ± 1.43 h after a 1 mg/kg intravenous administration to rats and bioavailability of 65.6 ± 4.3% for a 5 mg/kg subcutaneous dose, enabling its use in further in vitro and in vivo target validation. Citation Format: Allison Drew, Lorna Mitchell, Nathalie Rioux, Kerren Swinger, Scott Ribich, Suzanne Jacques-O9hagan, Trupti Lingaraj, Tim Wigle, Tom Riera, Richard Chesworth, Jesse Smith. Identification of the first small molecule PRMT6 inhibitor tool compound. [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 5376. doi:10.1158/1538-7445.AM2015-5376

Abstract 2437: Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates

Co-activator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers including AML (1) and breast (2), prostate (3), lung (4) and colorectal (5) carcinomas. CARM1 is known to methylate H3 histones and non-histone substrates including p300/CBP, AIB1/SRC-3 and PABP1 (6). To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors (7), but no ternary structures with nucleotide and peptide substrate have been reported. Here, the crystal structures of human CARM1 with the SAM mimic sinefungin (SFG) and three different peptide sequences from histone H3 and PAPB1 are presented, and both non-methylated and singly-methylated arginine residues are exemplified. Extensive interactions are seen with residues Glu266, Glu257, and His414 and the substrate arginine side chain. Two key hydrogen bonds are made by the side chain of Asn161 with the backbone carbonyl of the P’1 peptide residue and the backbone NH of the P’3 peptide residue. The carbonyl of the P1 peptide residue engages the protein through water mediated hydrogen bonds. These structures show how the CARM1 binding site is capable of accommodating a variety of peptide sequences. Comparisons to known CARM1 complexes with small molecules provide additional insights into inhibitor design. 1. Vu, L. P. et al, PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex. Cell Rep 2013, 5 (6), 1625-38. 2. Al-Dhaheri, M. et al, CARM1 is an important determinant of ERalpha-dependent breast cancer cell differentiation and proliferation in breast cancer cells. Cancer Res 2011, 71 (6), 2118-28. 3. Kim, Y. R. et al, Differential CARM1 expression in prostate and colorectal cancers. BMC Cancer 2010, 10, 197. 4. Elakoum, R. et al, CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features. Biochimie 2014, 97, 210-8. 5. Ou, C. Y. et al, A coactivator role of CARM1 in the dysregulation of beta-catenin activity in colorectal cancer cell growth and gene expression. Mol Cancer Res 2011, 9 (5), 660-70. 6. Bedford M.T.; Clarke S.G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33, 1-13. 7. Sack, J.S et al, Structural basis for CARM1 inhibition by indole and pyrazole inhibitors. Biochem J 2011, 436, 331-9. Citation Format: Ann Boriack-Sjodin, Lei Jin, Suzanne L. Jacques, Allison Drew, Margaret Porter Scott, Scott Ribich, Oscar Moradei. Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates. [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 2437. doi:10.1158/1538-7445.AM2015-2437