Targeting Activated Signaling Pathways for the Treatment of IKZF1-Deleted B Lymphoblastic Leukemia

In B lymphoblastic leukemia (B-ALL), genome-wide association studies have revealed that deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are present in nearly 30% of patients. These lesions are most prevalent in high-risk subsets, including greater than 60% of patients with Philadelphia chromosome positive (Ph+) and Ph-like ALL. IKZF1 deletions are associated with an increased risk of relapse, therapy resistance, and inferior survival. It is therefore imperative to devise new treatment strategies for this poor-prognosis subset of patients.

To this regard, using novel CRISPR-Cas9 genome editing strategies, we developed a series of human B-ALL cell lines with IKZF1 deletions. These robust model systems have allowed us to investigate the underlying biology of IKZF1-deleted B-ALL. Our studies have thus far shown that IKZF1 deletion results in a stem cell-like gene expression profile, enhanced bone marrow homing and engraftment, and cell-intrinsic chemoresistance, consistent with the relapsing disease phenotype observed in affected patients. We are using these model systems to explore possible mechanisms of chemoresistance and delineate new strategies to improve response to therapy.

Global gene expression analysis of the engineered Nalm-6 IKZF1-deleted cells by RNA-seq revealed potential therapeutic vulnerabilities. IKZF1-deleted cells are characterized by increased activation of the JAK/STAT pathway with overexpression of JAK1, JAK3, STAT3, and STAT5. Aberrant activation of this pro-survival, anti-apoptosis pathway is associated with poor-prognosis leukemia; thus, we postulated this is a contributor to the chemoresistance inherent to IKZF1-deleted B-ALL. We explored the therapeutic potential of targeting the JAK/STAT pathway by treating IKZF1-deleted cells with selective inhibitors of JAK1/3 (tofacitinib) and STAT3 (MM-206) and calculated the IC₅₀ by Annexin V/7-AAD double-negative population after 48 to 72 hours of treatment. The IKZF1 wild-type cells were sensitive to both compounds, suggesting activated JAK/STAT signaling is critical to cell survival. In comparison, the IKZF1-deleted cells were relatively resistant to both compounds (MM-206 IC₅₀ : 5.6 µM vs. 8.2 µM, p < 0.001; tofacitinib IC₅₀ : 43 nM vs. 55 nM, p = 0.05) similar to the relative resistance to ABL1-tyrosine kinase inhibition observed in Ph+ B-ALL cells with loss of function IKZF1 mutations. However, we postulated that inhibition of the JAK/STAT pathway could still augment the effects of standard chemotherapy. Indeed, whereas IKZF1-deleted Nalm-6 cells are highly resistant to glucocorticoid chemotherapy alone, when the cells were also treated with sub-IC₅₀ levels of MM-206, we observed a significant re-sensitization to dexamethasone-induced apoptosis. A similar pattern of re-sensitization was seen with the combination of sub-IC₅₀ MM-206 and vincristine treatment.

Additionally, our gene expression analysis of the IKZF1-deleted Nalm-6 cells revealed significantly increased expression of the receptor tyrosine kinase, FLT3. Overexpression was confirmed at the protein level by flow cytometry for cell-surface FLT3. We treated our engineered cell lines with the potent and selective FLT3 inhibitor quizartinib and again found that the IKZF1-deleted cells were relatively resistant compared to the wild type cells (IC₅₀ : 240 nM vs. 282 nM, p < 0.01). Postulating that parallel activation of the JAK/STAT pathway may contribute to this resistance, we treated our cells with pacritinib, a combined JAK/FLT3 inhibitor. The IKZF1-deleted cells were as sensitive to this compound as the wild type cells, suggesting dual targeting of FLT3 and JAK may be efficacious for the treatment of IKZF1-deleted B-ALL.

Our data support that IKZF1-deleted B-ALL is an aggressive disease characterized by cell-intrinsic chemoresistance. We found that loss of IKAROS amplifies pro-survival, anti-apoptotic signaling pathways, a likely contributing mechanism to chemoresistance. IKZF1 deletion confers relative resistance to targeted inhibitors of these pathways. However, the combined JAK/FLT3 inhibition exhibits therapeutic efficacy. Additionally, the combination of a JAK/STAT pathway inhibitor with conventional chemotherapy including dexamethasone and vincristine may be a promising strategy to overcome the chemoresistance inherent to this poor-prognosis subset of B-ALL.