Pb1791: slfn11 regulates multiple pathways that are important for treatment response in aml

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Despite recent advances in treatment for acute myeloid leukemia (AML), AML remains a deadly disease, with approximately 70% of patients succumbing to the disease within five years of diagnosis (SEER database 2021). Intensive induction chemotherapy carries a risk of morbidity and mortality. Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DAC) are now frequently used in AML treatment as less intensive treatment options. Thus, biomarkers to predict response and novel agents in AML are needed. We found a significant association between AML patient survival and high expression of Schlafen11 (SLFN11), a key effector of the DNA replication stress response. SLFN11 has been shown to be associated with response to certain chemotherapy agents in some solid tumors in vitro and in vivo (Conteduca et. al. JCO 2019; Shee et. al. PLoS One 2019), but its importance in leukemia has not been defined. Aims: We will determine whether SLFN11 can be used as a predictive biomarker for treatment response to DNA-damaging agents and/or HMAs in AML. We will also test whether targeting pathways upregulated in SLFN11-low cells could lead to novel combination therapies. Methods: The human leukemia cell lines HEL and U937 cells were used for these studies. U937- and HEL-SLFN11 knockout (SLFN11KO) cells were generated using CRISPR-Cas9 gene editing technology. Cell viability was assessed by WST-1 reagent assay. SLFN11 and other protein levels were measured by Western blotting. Results: U937- and HEL-SLFN11KO cells were incubated with increasing concentrations of cytarabine (AraC). Both U937- and HEL-SLFN11KO cells were more resistant to AraC than their respective control cells. To examine the mechanism of resistance, SLFN11KO and control cells were treated with AraC for up to 8 hrs, and lysates were subjected to immunoblotting for DNA damage proteins. There were higher baseline levels of multiple DNA damage-related proteins in the SLFN11KO cells. There was also prolonged activation of the DNA damage pathway in U937-SLFN11KO cells, without a concomitant increase in γH2AX (a marker of DNA damage and double strand breaks). Because U937-SLFN11KO cells demonstrated a more pronounced activation of the ATR/Chk1 pathway in response to AraC, we subjected U937-SLFN11KO and control cells to increasing concentrations of AraC with a small dose (0.3 μM) of the ATR inhibitor VE822. Notably, ATR inhibition restored sensitivity of SLFN11KO cells to AraC. In contrast, SLFN11KO cells and control cells were equally sensitive to AZA and DAC, suggesting that HMAs may be preferable to AraC for patients with SLFN11-low AML. To find additional targets in SLFN11-low AML, we performed RNA-seq on U937-SLFN11KO cells and noted that two genes (PDE7B and AKAP7) in the cyclic AMP pathway were upregulated. Notably, higher expression of these genes is associated with worse overall survival in AML patients, suggesting that targeting the cyclic AMP pathway may represent a new therapeutic strategy in AML. Summary/Conclusion: Low SLFN11 levels predict poor response to standard chemotherapy agents (i.e. AraC) in AML cells, suggesting that SLFN11 may prove to be an important predictive biomarker for AML patients. In addition, SLFN11-low AML cells have an increased reliance on DNA repair pathways, and inhibition of DNA repair proteins such as ATR may enhance responses to AraC in these cells. Finally, SLFN11KO cells overexpress PDE7B and AKAP7. This raises the possibility of patient selection based on SLFN11 levels for specific combination approaches for the treatment of AML. Keywords: Cytarabine, AML, Resistance, Acute myeloid leukemia