Rational Polypharmacological Targeting of FLT3, JAK2, ABL and ERK1 By Pluripotin Suppresses the Adaptive Resistance to FLT3 Inhibitors in AML

Background: Despite significant advancement in developing FLT3 inhibitors, treatment failure is common due to emergence of resistance. Most patients developed resistance within short duration even on continued TKI therapy. Both type-I (gilteritinib and crenolanib) and type-II (quizartinib and sorafenib) FLT3 inhibitors have been evaluated in patients. Regardless of their selectivity and mode of inhibition, resistance emerged. Nonetheless, mechanisms driving resistance partly differ between type-I and type-II inhibitors. For instance, resistance to type-II inhibitors is predominately mediated by the on-target selection of resistant mutations in the FLT3 kinase domain, while resistance to type-I inhibitors is principally driven by off-target activation of RAS-MAPK, BCR-ABL, JAK2/JAK1 signaling. A minority of resistant patients showed the emergence of mutations in genes regulating metabolism and transcription. However, its relevance in conferring resistance has not been evaluated. Besides, like other TKI treated malignancies, gatekeeper mutation (F691L) conferred resistance to both type-I and type-II FLT3 inhibitors and poses a significant clinical challenge. It is not clear why on-target acquired resistance is more frequent with type-II inhibitors in comparison to type-I inhibitors where adaptive resistance to compensatory signaling is more common. It is likely possible that stabilization of active kinase conformation by type-I inhibitor is, nonetheless, enzymatically active but its non-enzymatic function (signaling scaffold) drives the adaptation to alternate survival pathways. Nevertheless, these studies informed that FLT3 resistant variants could be suppressed by switching to next-generation FLT3 inhibitors or dose escalation as demonstrated in managing the TKI resistance in chronic myeloid leukemia (CML). However, unlike CML, resistance conferred by off-target activation of MAPKs, BCR-ABL, and JAKs in AML remains a serious challenge.

Results: The persistence of residual disease is the root cause of resistance. Strategies aimed at greater front-line disease eradication and suppression of resistance are needed, most of which depends on further research into combination chemotherapy or to develop polypharmacological agents targeting FLT3, its resistant variants, BCR-ABL, JAK2, and MAPKs. To address this, we performed a cell-based screening to identify the small molecule inhibitor active against FLT3, BCR-ABL, JAK2, and MAPKs. Here we show that pluripotin (SC-1), an inhibitor of RASGAP, and MAPK3, potently inhibits the kinase activity of FLT3, BCR-ABL, and JAK2. Structural modeling studies revealed that it binds with inactive conformations of FLT3, JAK2, and ABL. It is an equipotent inhibitor to both FLT3^ITD and its most vexing resistant variant, the gatekeeper mutant F691L. Consequently, it potently suppressed the leukemic progression of TKI resistant primary AML in a preclinical mouse model, FLT3^ITD/ITD/Tet2^-/- mice develop robust leukemia and are resistant to FLT3 TKIs, e.g., Quizartinib and Gilteritinib, Fig 1. As expected, pluripotin treatment efficiently suppressed the adaptive resistance conferred by MAPK, BCR-ABL, and JAK2 signaling. As a proof of concept, we provide evidence that unique polypharmacology of pluripotin targeting FLT3, BCR-ABL, JAK2, and MAPK efficiently suppressed the leukemic progression in multiple preclinical mouse models as well as in mice engrafted with primary AML cells.

Conclusions: Our preclinical data suggest that up-front targeting of key signaling nodes driving adaptive resistance by polypharmacological agents provides durable response, which is not achieved by currently used FLT3 inhibitors. Altogether, these studies warrant clinical evaluation of pluripotin to suppress the adaptive resistance for durable response.