Emily Lee

Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-cell Acute Lymphoblastic Leukemia

Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease. Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity. Results: We first determined that both NOTCH1-mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice. Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012–24. ©2016 AACR. See related commentary by Carroll et al., p. 873

Antitumor synergy with SY-1425, a selective RARα agonist, and hypomethylating agents in retinoic acid receptor pathway activated models of acute myeloid leukemia

Acute myeloid leukemia (AML) and biologically related myelodysplastic syndrome (MDS) are hematologic malignancies with poor outcomes. While recent approvals of new targeted therapies have increased options for some patients, those unfit for intensive treatment have few options, with single agent hypomethylating agents (HMAs) remaining as standard of care. The retinoic acid receptor alpha (RARα) transcription factor, encoded by the RARA gene, plays a critical role in myeloid cells and shows dysregulation in a subset of AML and MDS tumors. We recently demonstrated that the selective RARα agonist SY-1425 (tamibarotene) had biologic and clinical activity in 43% of evaluable relapsed or refractory AML and higher-risk MDS patients with activation of the RARA pathway. In this study, we sought to determine whether HMAs and SY-1425 exerted synergistic antiproliferative effects in AML models of RARA pathway activation in vitro and in vivo. Addition of HMAs and SY-1425 to RARA-high or IRF8-high, but not RARAlow, AML cell lines resulted in synergistic antiproliferative effects supported by evidence of DNA damage and apoptosis to a far greater extent than either agent alone. Studies in a patient-derived xenograft mouse model also demonstrated deeper and more durable responses with the combination than either agent alone. Furthermore, preclinical testing of various regimens determined that treating with azacitidine for one week followed by treatment with SY-1425 for three weeks maximized tumor suppression and tolerability. These findings directly support the ongoing clinical study of SY-1425 in combination with azacitidine. Both AML and MDS arise, in part, due to genetic alterations in transcription factors (i.e., RUNX1, NPM1) and epigenetic modifying genes (i.e., MLL, DNMT3A) leading to inactivation of tumor suppressor genes, thus enabling proliferation of immature cells. Alterations in DNA methyltransferases (DNMTs) specifically result in DNA hypermethylation which contributes to gene silencing through promoter inactivation, and can be targeted by HMAs that mimic native nucleoside residues and incorporate into DNA. Once incorporated, HMAs are recognized by DNMT1 as a cytosine, however this interaction creates an irreversible DNA-DNMT1 adduct that requires DNA damage repair to resolve. This then results in loss of DNMT1, as the DNA-protein adduct is degraded by the DNA damage response pathway. After loss of

Abstract 1511: AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner

Prior studies have shown that the RARA gene is associated with a super-enhancer (SE) and has upregulated mRNA expression in a subset of AML patients. Furthermore, this has been found to confer increased sensitivity to SY-1425, a potent and selective RARα agonist. We sought to better characterize the cell state and transcription factor circuitry in these RARA-high AML cells. Clustering of 62 primary AML patient samples based on their genome wide SE maps identified six discrete clusters. RARA-high patients partitioned principally into cluster 2, and to a lesser extent 1, suggesting that RARA upregulation is associated with a specific transcription factor (TF) network and cell state. To start unraveling the TF circuitry in the RARA-high cluster, we investigated which other TFs were SE associated with clusters 1 and 2. In particular, interferon regulatory factor 8 (IRF8) was found to be strongly associated with clusters 1 and 2 by SE and mRNA expression, similar to RARA. Moreover, the expression of both genes is correlated in primary patient samples. IRF8 is involved in interferon signaling and previous studies have shown crosstalk between interferon and retinoic acid signaling. Furthermore, aberrant IRF8 pathway signaling is implicated in AML and CML pathogenesis. The tight clustering of RARA and IRF8 in patient subgroups defined by genome wide enhancer maps suggests RARα and IRF8 may form an integrated transcriptional circuit. Indeed, treatment with SY-1425 was found to strongly induce interferon-like gene expression changes in AML cells with high RARA or IRF8 levels, including the tumor suppressive IFN responsive gene IRF1. While RARA-high AML cell line models have been previously shown to respond to SY-1425, we found that models with high IRF8 expression and low levels of RARA were also found to respond to SY-1425. Such IRF8-high, RARA-low AML cell lines showed activation of similar transcriptional pathways as RARA-high cell lines in response to SY-1425 based on GSEA. IRF8-high AML also had comparable low nM EC50 anti-proliferative effects following SY-1425 treatment. In addition, SY-1425 was found to elicit differentiation in both RARA-high and IRF8-high AML cell lines based on flow cytometry. While RARA and IRF8 expression appear correlated, this data suggests that IRF8 levels may predict for sensitivity to SY-1425 in addition to RARA levels, particularly in cases of AML with high IRF8 expression but low RARA levels. Insights derived from enhancer analysis, transcriptional profiling and differentiation response in preclinical models support the recently initiated Phase 2 trial of SY-1425 (NCT02807558) in which we are evaluating the SE based patient selection strategies and gene circuitry derived pharmacodynamics clinical measurements, including differentiation markers, in patients with AML and MDS. Citation Format: Michael R. McKeown, Matthew L. Eaton, Chris Fiore, Emily Lee, Katie Austgen, Darren Smith, M. Ryan Corces, Ravindra Majeti, Christian C. Fritz. AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner [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 1511. doi:10.1158/1538-7445.AM2017-1511

Abstract 3085: SY-1425 (tamibarotene), a selective RARα agonist, shows synergistic anti-tumor activity with hypomethylating agents in a biomarker selected subset of AML

In patients with acute myeloid leukemia (AML) (≥ 60 years) and myelodysplastic syndrome (MDS), the use of hypomethylating agents (HMAs) may extend survival, but cure rates are very low and new treatment approaches are needed. HMAs, such as azacitidine, work by inhibiting DNMT1, leading to depletion of DNA methylation in the tumor cells. Hypomethylation, in turn, leads to the re-expression of genes associated with differentiation and growth arrest. We have recently explored the potent and selective RARα agonist SY-1425 in a genomically defined subset of AML. SY-1425 binds to RARα and causes a transition from repression to strong activation of target genes, thus reprogramming the tumor cells toward terminal maturation in RARA-high AML models, supporting our recently initiated Phase 2 trial in a biomarker-selected subset of AML and MDS (NCT02807558). Based on potential mechanistic synergy, we evaluated SY-1425 in combination with HMAs and identified a synergistic anti-proliferative effect. In RARA-high AML cell lines, but not RARA-low, the combination of SY-1425 with either azacitidine or decitabine showed synergistic anti-proliferative effects on the cells, with combination indices less than 0.5 over a range of concentrations from 0.01 to 100nM of SY-1425 and 0.1 to 1µM of HMAs. SY-1425 and azacitidine were also co-administered to a disseminated patient-derived xenograft (PDX) mouse model of RARA-high AML. The combination demonstrated superior reduction of tumor burden vs either therapy alone, leading to deeper and more durable responses with less than 1% detectable tumor burden. A follow-up study in the RARA-high PDX model investigated different treatment schedules of SY-1425 and azacitidine over a period of 56 days, supporting a regimen that maximizes anti-tumor activity and tolerability. Mechanistic studies using RNA-seq and ChIP-seq in AML cell line models have revealed that while azacitidine had only moderate suppressive or activating effects over a broad set of genes, the addition of SY-1425 in RARA-high models resulted in strong and specific induction of genes bound by RARα. It is hypothesized that azacitidine acts to prime the tumor cells for reprogramming by SY-1425. The loss of methyl-cytosine residues following azacitidine treatment lowers the barrier to SY-1425 mediated gene induction. It was observed that the two agents work cooperatively to promote terminal differentiation and decrease proliferation of the AML tumor cells, with the potential for increased clinical benefit in a subset of AML defined by a RARA super-enhancer. Based on the largely non-overlapping clinical toxicity profiles of azacitidine and SY-1425, supported by the observed tolerability of the combination in preclinical models, these findings provide a strong rationale for a planned study of this combination in biomarker selected, newly diagnosed AML patients. Citation Format: Michael R. McKeown, Emily Lee, Chris Fiore, Matthew L. Eaton, Christian C. Fritz, Eric Olson. SY-1425 (tamibarotene), a selective RARα agonist, shows synergistic anti-tumor activity with hypomethylating agents in a biomarker selected subset of AML [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 3085. doi:10.1158/1538-7445.AM2017-3085

Abstract P6-11-18: A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment

Endocrine-resistance remains a major challenge for treatment of breast cancer. Multiple mechanisms for endocrine resistance have been proposed, including altered expression of ER co-regulators such as Retinoic Acid Receptor Alpha (RARα). Furthermore, crosstalk between estradiol and RA signaling is known and upregulation of RARα has been observed in tamoxifen resistance. We propose a novel treatment paradigm for a newly-defined subset of HR+ patients based on our discovery of a super-enhancer (SE) associated with the RARA locus. SEs are large, highly active chromatin regions that pinpoint cancer vulnerabilities. The RARA SE-identified vulnerability can be targeted using the potent, selective, and metabolically stable RARα agonist SY-1425 (tamibarotene). SY-1425 is approved in Japan to treat Acute Promyelocytic Leukemia, has a well-established efficacy and safety profile, and may enhance response to hormonal therapy (HT) in this newly-defined subset of HR+ patients potentially delaying the need for alternate treatment. Tumor samples from 42 breast cancer patients were analyzed across a range of molecular subtypes. We identified an SE linked to the RARA gene in 54.5% of the hormone positive patient samples. RARA SEs predicted sensitivity to SY-1425 in 12 breast cancer cell lines confirming their functional role, and showed a correlation with RARA gene expression. A panel of 37 breast cancer cell lines was tested for SY-1425 anti-proliferative activity and gene expression levels, and identified RARA as the single best predictor of response. Proliferation of RARA-high cells was inhibited by SY-1425 with low nanomolar EC50s. Transcriptional profiling was performed on 4 HR+ and 3 HER2+/HR- breast cancer cell lines and analyzed by GSEA to examine the molecular response to SY-1425. Signatures for growth including E2F, MYC, DNA replication, and cell cycle were significantly downregulated while retinol metabolism and luminal signaling were upregulated. Estrogen signaling was also significantly altered by SY-1425, supporting known crosstalk between RARα and ER. Consistent with differentiation, CYP26A1 and VE-Cadherin were induced and Actin and Ki67 were diminished at relevant concentrations of SY-1425 and could serve as pharmacodynamic markers of response. To test responses to SY-1425 in vivo , two cell line-derived models and two patient-derived breast cancer models (one RARA-high, and one RARA-low each) were treated with SY-1425. SY-1425 inhibited tumor growth in the RARA-high models, but not the RARA-low models (43% versus 0% TGI). Consistent with the observed changes in transcription, SY-1425 in combination with tamoxifen synergistically inhibited proliferation of RARA-high breast cancer cell lines. Although a few clinical studies have investigated the use of ATRA in HR+ breast cancer without success, our results suggest that patient selection based on the RARA SE may predict which HR+ breast cancer patients could derive benefit by adding an RARα agonist to HT. The potential to prolong or increase the clinical effect of anti-estrogen therapy with SY-1425, which has improved potency, selectivity, and PK stability versus ATRA, would be an attractive strategy to explore. Citation Format: McKeown MR, Fiore C, Lee E, Eaton ML, Orlando D, Guenther MG, Collins C, Chen MW, Fritz CC, di Tomaso E. A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-18.