Abstract
Abstract 672
Long-term disease-free survival is achieved in over 80% of children with B-cell acute lymphoblastic leukemia (B-ALL) but only 40% of adults. Particular genomic alterations in B-ALL, including rearrangements of MLL and the cytokine receptor subunit CRLF2, confer a poor prognosis in both children and adults. In addition, current therapies for B-ALL are associated with significant short- and long-term toxicities, highlighting the critical need for new therapeutics. The novel compound JQ1 inhibits the BET class of human bromodomain proteins from mediating the assembly of macromolecular protein complexes that are required for transcriptional activation and polymerase elongation. In hematologic and epithelial tumors, JQ1 can downregulate the expression of c-MYC and thereby suppress malignant growth and survival. We investigated the therapeutic potential of JQ1 across multiple genetically-defined subsets of B-ALL. JQ1 potently induced apoptotic cell death (IC50∼30–300 nM) in all B-ALL cell lines (697, CEMO-1 NALM-6, MHH-CALL4, MUTZ-5, Reh, RS4;11, SEMK2) studied. Among the most sensitive lines (IC50<50 nM) were MUTZ-5 and MHH-CALL4, which both harbor IGH@-CRLF2 translocations as well as activating mutations in JAnus Kinase 2 (JAK2). CRLF2 heterodimerizes with the IL7 receptor (IL7R) subunit in response to thymic stromal lymphopoietin, which induces JAK/STAT, MAP kinase and AKT signaling. To identify mechanisms through which JQ1 induces cell death in MUTZ-5 and MHH-CALL4 cells, we quantified transcript and protein levels for relevant targets in the presence of JQ1 500 nM or vehicle (DMSO). Chromatin immunoprecipitation was also performed with antibodies against BRD4 followed by PCR to determine the effects of JQ1 on BRD4 binding at relevant promoters. As previously observed, JQ1 induced the downregulation of MYC mRNA, loss of BRD4 at the MYC promoter, and reduced the expression of c-Myc target genes. Immunoblotting with phospho-specific antibodies demonstrated almost complete loss of JAK2 and STAT5 phosphorylation in cells treated with JQ1, suggesting that JQ1 also blocks signaling downstream of CRLF2/IL7R. While the levels of CRLF2 mRNA were unaffected by JQ1 in MUTZ-5 and MHH-CALL4 cells, JQ1 markedly downregulated IL7R mRNA and depleted BRD4 from the IL7R promoter in both lines. The reduction in IL7R mRNA levels led to dramatic decreases in IL7R surface expression. Genome-wide expression profiling demonstrated a highly restricted effect of JQ1, with IL7R and MYC being the 7th and 23rd most downregulated genes, respectively. In fact, IL7R was the only cytokine receptor in both CRLF2-rearranged B-ALL lines that was significantly downregulated by JQ1 treatment. In addition, JQ1 potently reduced IL7R mRNA across other B-ALL cell lines with diverse cytogenetics. To determine whether JQ1 could suppress the growth of human B-ALL in vivo, we xenografted a human CRLF2-rearranged B-ALL primary sample into Nod.SCID.IL2RG−/− mice. Upon the development of >30% bone marrow involvement by human CD45+/CRLF2+ B-ALL cells, mice were randomized to receive JQ1 (50mg/kg intraperitoneally daily) or vehicle (DMSO). After 5 days of treatment, sentinel mice were sacrificed for pharmacodynamic endpoints. Spleens from mice treated with JQ1 had markedly reduced c-Myc expression and STAT5 phosphorylation compared with spleens from vehicle-treated mice. In survival cohorts (n=9 per arm), treatment with JQ1 significantly prolonged overall survival (p=0.0002) compared with vehicle. These results demonstrate that BET bromodomain inhibition is a promising therapeutic strategy for patients with B-ALL, including subsets with high-risk cytogenetics. Moreover, the surprising finding that JQ1 also targets IL7R expression suggests that bromodomain inhibitors may be especially useful in malignant and nonmalignant disorders dependent on IL7R.
Bradner:Tensha Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SHAPE Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weinstock:Novartis: Consultancy, Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
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