ALBERT

Description: Highly aggressive acute myeloid leukemia (AML) is the most common cause of leukemia-related deaths in the United States. The development of new treatments for acute leukemias that would increase durable responses and improve poor survival rates with manageable toxicity is imperative. Certain cancers, including acute lymphoblastic leukemia (ALL), are dependent on oncogenic transcription factors and are particularly sensitive to selective CDK7 inhibitors (Kwiatkowski et al., 2014; Chipumuro et al., 2014; Christensen 2014). AML tumors harbor fewer somatic mutations compared to solid tumors and many of these mutations affect proteins critical to gene control, potentially conferring a unique vulnerability to the inhibition of transcriptional processes. CDK7 is a transcriptional kinase that phosphorylates the C-terminal domain of RNA polymerase II, enabling processive transcription. CDK7 has previously been implicated in the regulation of super-enhancer (SE) mediated circuitry. Here, we report preclinical in vitro and in vivo data in AML with a first-in-class CDK7 inhibitor, SY-351, that covalently targets a cysteine outside the kinase domain, resulting in sustained, highly selective inhibition. SY-351 shows potent inhibition of CDK7 in biochemical (Ki = 62 nM and Kinact = 11.3 hr-1) and cell-based (EC50 = 4.7 nM HL-60) assays and, when profiled against nearly 400 other kinases, exhibits limited off-target inhibition. In in vitro studies, potent and selective inhibition of CDK7 with SY-351 results in rapid induction of apoptosis in AML cell lines, while inducing little to no cell death in normal immortalized cell lines at comparable levels of target engagement. In vivo, CDK7 inhibition did not show myelosupression that has previously limited the clinical development of pan CDK inhibitors. Orthotopic AML and ALL patient-derived xenotransplants (PDX), treated with SY-351 results in complete responses (CR) where the clearance of blasts from blood and marrow was achieved without body weight changes or cell death in normal hematopoietic cells. These effects led to increased survival in mouse PDX models, and the response was sustained following removal of the drug. In summary, we describe a first-in-class CDK7 inhibitor that is potent and selective, well tolerated at efficacious doses and that leads to durable, complete responses in PDX models of AML and ALL. These data support the further exploration of this class of compounds for clinical development. Disclosures Ren: Syros Pharmaceuticals: Employment, Equity Ownership. Brown:Syros Pharmaceuticals: Employment, Equity Ownership. Hu:Syros Pharmaceuticals: Employment, Equity Ownership. Lopez:Syros Pharmaceuticals: Employment, Equity Ownership. Miljovska:Syros Pharmaceuticals: Employment, Equity Ownership. Schmidt:Syros Pharmaceuticals: Employment, Equity Ownership. Bradley:Syros Pharmaceuticals: Employment, Equity Ownership. Sprott:Sprott Biotech LLC: Other: Spouse Kam M. Sprott is a consultant; Syros Pharmaceuticals: Employment, Equity Ownership. Olson:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Choi:Syros Pharmaceuticals: Employment, Equity Ownership.

Description: SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

Description: Retinoic acid receptor alpha (RARα) regulates myeloid differentiation and proliferation through the regulation of specific sets of genes. When unbound by a ligand, RARα is a repressive transcription factor while in its ligand-bound state it functions as a transcriptional activator. Previously, blast cells from a subset of individuals with non-APL AML were found to have a super-enhancer (SE), as revealed by H3K27 acetyl ChIP-Seq, associated with the RARA locus (hereafter called RARA-high), suggesting that tumor cell proliferation may have a dependency on RARA that can be exploited for therapeutic benefit. SEs are exceptionally large, highly active chromatin regions that are densely occupied by transcription factors and have been implicated in oncogene expression. Indeed, RARA-high non-APL AML cell lines showed 〉1000-fold increased sensitivity compared to RARA-low cells to the potent and selective RARα agonist SY-1425 (tamibarotene) as well as efficacy in non-APL AML patient derived xenograft models with a dependency on RARA. Since RARα is a transcription factor and the direct target of SY-1425, we investigated the change SY-1425 had on the transcriptional program of non-APL AML cell lines and the mechanism underlying those changes. Expression profiling on a panel of AML cell lines revealed that RARA-high AML cell lines had profound transcriptional changes in response to SY-1425, with 437 genes significantly changed, while RARA-low cell lines did not show significant gene expression changes. Gene set enrichment analysis (GSEA) of three RARA-high AML cell lines revealed that the genes upregulated by SY-1425 in the RARA-high cells are associated with immune signaling, interferon induction, protein secretion, and pathways associated with complement, MHC and integrin functions, all pathways indicative of more differentiated blood cells. Signatures downregulated by SY-1425 include MYC target genes. These findings are consistent with SY-1425 increasing the expression of genes involved in differentiation and decreasing those involved in proliferation. Genome-wide ChIP-Seq analysis revealed an increase in H3K27 acetylation at loci found to have strong RARα peaks as well as increased expression of those genes upon treatment with SY-1425. Together, these data support a model in which RARα binding nucleates functional enhancers in response to SY-1425 thereby upregulating proximal target gene expression and promotion of differentiation. The gene expression and epigenomic responses of RARA-high AML cell lines to SY-1425 were found to be similar to the responses of an APL cell line (NB-4) to retinoids or SY-1425. Gene sets identified in response to either retinoid treatment or genetic perturbation, such as forced expression or RAR-fusions or knockdown, matched the gene sets identified in RARA-high AML cell lines. Furthermore, the quantitative response of both NB-4 and RARA-high AML cell lines to SY-1425 was found to be similar. Across the genome, RARα binding was highly conserved between NB-4 and RARA-high AML cell lines with less overlap with the RARA-low cell lines. For example, the transcriptional and H3K27 acetylation alterations at the known PML-RARα target gene TGM2 following retinoid treatment was similar in NB-4 and the RARA- high cell lines. This locus also had a strong RARα binding site that is conserved among the cell lines and co-localized with a strong H3K27 acetylation peak. Consistent with the pattern of occupancy of RARα on the genome, the transcriptional response of the RARA enhancer-high cell lines to SY-1425 treatment was similar to the response of APL ex-vivo patient samples to retinoic acid treatment. These data support a model of a common biological response to retinoids between cells with the RARA-PML translocation in APL and cells with the RARA SE in AML. The mechanistic studies described here support the therapeutic potential of SY-1425 in myeloid leukemia patients who have a SE associated with RARA. A biomarker directed clinical trial of SY-1425, a potent and selective RARα agonist, in a subset of AML and MDS patients with an altered RARA locus (clinicaltrials.gov, NCT02807558) is supported by these data. Disclosures Fiore: Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.