Selection of RARA-Positive Newly Diagnosed Unfit AML Patients with Elevated RARA Gene Expression Enriches for Features Associated with Primary Resistance to Venetoclax and Clinical Response to SY-1425, a Potent and Selective RARα Agonist, Plus Azacitidine

Introduction: Super-enhancer (SE) mapping in non-APL AML patient (pt) blasts identified RARα as a novel therapeutic target in approximately 30% of pts, who have elevated RARA gene expression. It was observed that the enhancer profile of this novel pt segment, where RARA expression was elevated, overlapped with the profile of mature monocytes (McKeown, 2017). Recently, several reports describe AML with monocytic features associated with resistance to venetoclax (Ven), a BCL-2 inhibitor that has emerged as a standard of care for treatment of pts with newly diagnosed (ND) unfit AML in combination with hypomethylating agents (HMAs) (Zhang, 2018; Kuusanmäki, 2019; Pei, 2020). Approximately one-third of pts do not respond to Ven plus HMAs including azacitidine (Aza) (DiNardo, 2019 and 2020), highlighting a continuing significant unmet need in ND unfit AML. SY-1425, a potent and selective RARα agonist, is in development for non-APL AML in combination with Aza and has demonstrated clinical activity with high rates of complete remission (CR) and deep CRs in RARA-positive (RARA+) ND unfit AML (DeBotton, 2019). Based on the overlap of monocytic features with RARA gene expression, we evaluated clinical samples of pts treated with SY-1425 plus Aza to correlate features of Ven resistance with the RARA biomarker and with clinical response to SY-1425 plus Aza.

Methods: RARA gene expression in non-APL AML was evaluated in the TCGA and Beat AML RNA-seq datasets. AUC of cell viability curves were used to evaluate ex vivo sensitivity to compounds, including Ven, in the Beat AML dataset. A monocytic expression signature (MES) was developed using the expression of monocytic and primitive RNA markers in the TCGA dataset to analyze the monocytic phenotype. The MES used a logistic regression model with lasso regularization to distinguish FAB M4/5 (monocytic) from FAB M0/1/2 (primitive) using 10-fold cross-validation with 85% sensitivity and 80% specificity. The MES was then applied to the RNA-seq datasets from Beat AML and AML blasts from ND unfit AML pts in the ongoing SY-1425 plus Aza trial (NCT02807558), in which RARA+ pts were determined by an RT-qPCR based biomarker clinical trial assay (CTA). The MES, RARA expression, and Ven resistance-associated features were compared using Spearman's rho correlation; the association of the MES with the RARA biomarker and with IWG clinical responses in SY-1425 plus Aza treated pts was evaluated.

Results: Analysis of RNA-seq in TCGA non-APL AML pts demonstrated higher RARA expression in monocytic AML (FAB M4/M5) than primitive AML (FAB M0/M1/M2) (p<10^-7, t-test). TCGA and Beat AML datasets also demonstrated that RARA expression was associated with the MES (rho=0.6 and 0.58), with approximately 70% of RARA-high pts across both databases having a high MES.

We further elucidated the relationships of RARA expression, AML monocytic phenotypes, and Ven resistance. Of 121 inhibitors tested ex vivo in primary Beat AML pt samples, Ven was the inhibitor most associated with treatment resistance in RARA+ vs. RARA- samples. Additionally, MES (rho=0.58), RARA (rho=0.48) and BCL-2 expression (rho=-0.49) had similar magnitude of association with ex vivo Ven resistance, with RARA+ samples showing much lower ex vivo sensitivity to Ven than RARA- samples (p=3×10^-8). In 12 AML pt samples (Pei, 2020) treated with Ven ± Aza ex vivo, RARA expression was higher in the monocytic leukemia stem cells resistant to Ven ± Aza (p=0.005).

To evaluate whether the RARA+ ND unfit AML pts in the ongoing SY-1425 plus Aza clinical trial were enriched for the monocytic phenotype of Ven resistance, RNA-seq was performed on enrolled pt AML blasts. Among 51 treated pts, 86% (19/22) of RARA+ and 83% (24/29) of RARA- pts yielded RNA-seq results. RARA+ pts were more monocytic than RARA- pts, as demonstrated by higher MES (p=0.002), with higher MCL-1 (p=0.001), and lower BCL-2, CD34, and CD117 expression (p=0.03, 8×10^-6, 2×10^-4, respectively). In pts with the best IWG response of CR/CRi, RARA+ pts (n=10) had higher MES than RARA- pts (n=8) (p=0.008).

Conclusion: In ND unfit AML, RARA+ pts, including those with clinical responses to SY-1425 plus Aza, are enriched for monocytic features associated with resistance to Ven. SY-1425 plus Aza shows potential as a novel targeted regimen for the treatment of RARA+ ND unfit AML and warrants further development in this genomically defined subset of AML pts who may be resistant to upfront SOC therapy with Ven.