UCSF

Targeted therapy has achieved clinical success in many cancer patients whose diseases are driven by defined genomic lesions resulting in pathologically activated kinases. While mutations revealed by tumor sequencing can identify novel therapeutic targets, it is critical to distinguish between non-transforming “passenger” mutations and transforming “driver” mutations potentially amenable to therapeutic targeting. In contrast with oncogenic ABL1 fusions, point mutations in the ABL1 proto-oncogene have yet to be validated for transforming capacity and potential for therapeutic targeting. We assessed a number of ABL1 1b point mutants identified in clinical isolates and found that select mutations transformed Ba/F3 cells to growth factor independence and caused constitutive kinase activation. Employing an in vitro random mutagenesis screen, we prospectively identified additional activating mutants that could be vulnerable to currently available ABL1 inhibitors. All activating ABL1 1b mutants characterized in our study exhibited sensitivity to ATP-competitive tyrosine kinase inhibitors and relative resistance to allosteric inhibitors targeting the myristoyl-binding pocket regardless of proximity to the allosteric site. As the allosteric inhibitor ABL001 is currently undergoing clinical trial evaluation for treatment-refractory chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoid leukemia, we cloned these ABL1 1b-activating mutations into BCR-ABL1 and found that most, except Y469H and P918L, also caused significant resistance to allosteric inhibitors. Thus, we identified BCR-ABL1 mutations with the potential to promote clinical resistance to an emerging class of allosteric inhibitors. Finally, we assessed cDNA from a cohort of CML patients expressing a dominant BCR-ABL1 T315I mutation, and targeted sequencing of ABL1 1b revealed a heterozygous T315I mutation in one patient. Presently, CML patients who acquire resistance to ATP-competitive ABL1 TKIs are screened only for BCR-ABL1 mutations, but our finding that select mutations confer greater resistance in the context of ABL1 1b suggests drug-resistant patients should be assessed for mutations in both ABL1 1b and BCR-ABL1. Another challenge to developing effective therapies is that acquired drug resistance or toxicity from multi-kinase inhibition frequently limits the long-term efficacy of TKIs. Activating mutations of the FLT3 receptor tyrosine kinase are validated therapeutic targets in acute myeloid leukemia, but available FLT3 TKIs are susceptible to secondary resistance mutations and limited in clinical utility by off-target effects. We interrogated the role of dimerization in FLT3 activation to address whether inhibiting receptor dimerization could be a therapeutic target. Using FLT3 truncation constructs and phosphorylation site mutants, we unexpectedly found that the cytoplasmic domain of oncogenic FLT3 ITD is highly transforming and resistant to TKIs, likely through enhanced STAT5 signaling, implying the extracellular and/or transmembrane domains may have an autoinhibitory function. Thus, it will be important to evaluate the relative contributions of different FLT3 domains to activation, dimerization, and autoinhibition. A better understanding of cooperativity between modes of kinase activation and deregulation could aid in developing novel therapeutic strategies exploiting fundamental protein regulatory mechanisms.