Fedratinib and Venetoclax Have Synergistic Activity Against B-Cell Acute Lymphoblastic Leukemia in Vitro

Treatment of relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) remains a challenge particularly in patients who do not respond to chemotherapy or immunotherapy. While a subset of these patients can be cured with allogeneic bone marrow transplant, success of the transplant is contingent upon achieving a minimal residual disease negative complete remission. The objective of this study was to assess the efficacy of fedratinib, a semi selective JAK2 inhibitor and venetoclax, a selective BCL-2 inhibitor, on the B-ALL cell lines RS4;11, NALM-6, and SUP-B15. While select studies have examined the efficacy of these drugs on B-ALL as single agents, data is lacking on whether these drugs work optimally as single agents or in combination. Cell lines were treated with either vehicle (DMSO), single agent fedratinib or venetoclax, or the combination of both inhibitors for 48 and 72 hours. Fedratinib doses were tested from 150 nM to 1 uM and venetoclax doses were tested from 2.5 nM to 500 nM. Live/dead discrimination was performed using flow cytometry and a 72-hour MTS assay to assess proliferation. Drug synergy was calculated using CompuSyn software to generate combination index (CI) scores, with a CI score less than 1 reflecting synergy. RNA was isolated from treated leukemia cells and processed on a Nanostring nCounter platform to assess potential changes in gene expression. Results were then analyzed using Rosalind data processing software. In RS4;11, the combination of 5 nM venetoclax and 525 nM fedratinib significantly increased cell death compared to single agent venetoclax (p=0.0451) and fedratinib (p=0.0077). Furthermore, the combination significantly decreased leukemia cell proliferation compared to single agent fedratinib (p<0.0001). In NALM-6, the combination of 300 nM venetoclax and 300 nM fedratinib significantly increased cell death compared to single agent fedratinib (p=0.0103). Combination therapy also significantly decreased cell proliferation compared to single agent fedratinib (p=0.0157). With SUP-B15, the combination of 5 nM venetoclax and 750 nM fedratinib significantly increased leukemic cell death compared to single agent fedratinib (p=0.0081). Combination therapy also significantly decreased cell proliferation compared to both single agent venetoclax (p=0.0012) and fedratinib (p<0.0001). Synergy between venetoclax and fedratinib was seen among respective dose combinations for all three cell lines RS4;11 (CI=0.57258), NALM-6 (CI=0.67991), and SUP-B15 (CI=0.19162). Analysis of gene expression in RS4;11 cells treated with combination fedratinib and venetoclax identified a 3.53-fold decrease in NR4A1 expression (p=0.0001), an orphan nuclear receptor for hormones, and a 4.29-fold increase in SOCS2 expression (p=0.02), a negative regulator of cytokine signaling. Combination therapy with fedratinib and venetoclax has synergistic activity in multiple B-ALL cell lines, in part through regulation of hormone and cytokine signaling. Future studies will explore this combination therapy in vivo and identify signaling pathways impacted by JAK2 and BCL2 inhibition.