Abstract
Abstract 13
A large body of studies has proposed that leukemia initiating cells (LICs) are the culprit of leukemia relapse from conventional therapies. Like normal hematopoietic stem/progenitor cells (HSPCs), LICs are thought to reside in the bone marrow (BM) endothelial and/or endosteal niche. Although the full nature of LIC-niche interaction remains elusive, it has been postulated as a useful target for leukemia therapy based on a dual rationale: on one hand, the survival of LICs may depend upon interactions with specific niche, while on the other hand, chasing LICs out of the BM niche may drive quiescent LICs into active cell cycle, sensitizing them for conventional chemotherapy. Recent progresses in studying the PML tumor suppressor and the CXCR4-antagonist AMD3100 have provided strong support for such a rationale (Blood 113, 6215; Nature 453, 1072). The Rho GTPase family member Cdc42 is a central regulator of cell proliferation, adhesion, and migration by integrating signals from multiple cell surface receptors. Ablation of Cdc42 in HSPCs in a conditional knockout mouse model leads to massive egress of HSPCs from BM to the peripheral blood (PB), a phenotype attributable to deficiencies in HSPC adhesion, migration, and F-actin polymerization. We recently demonstrated that pharmacological targeting of Cdc42 by a small molecule Cdc42 activity-specific inhibitor (CASIN) transiently and specifically inhibits Cdc42 activity and mimics the Cdc42 knockout mobilization phenotype by suppressing HSPC adhesion, migration, and F-actin polymerization. CASIN appears to be active in this regard on both murine and human blood progenitors in vitro and in xenografted mice (Blood 112: 68a, Nature Biotechnology under revision). In the present studies, we hypothesize that pharmacological targeting of Cdc42 by CASIN is effective in mobilizing LICs from the BM niche, thus providing a new method for combinatory therapy against LICs. To test this hypothesis, we used MLL-AF9/N-Ras human AML cells (MA9/N-Ras), whereby the N-Ras G12D oncogene was introduced into MLL-AF9 transduced human CD34+ umbilical cord blood (HCB) cells. These cells grow vigorously in vitro independent of cytokine supplementation and induce AML readily in humanized NOD/SCID-SGM3 (SGM3) mice. Xenotransplant experiments confirmed that MA9/N-Ras cells are clonal inducers of leukemia with the property of LICs, as animals transplanted with either bulk cultures or with single cell derived cultures succumbed to AML with similar latencies. In MA9/N-Ras cells CASIN effectively inhibited downstream effectors of Cdc42 such as p-PAK, p-MLC and p-FAK in a dose-dependent manner. In the SGM3 mouse xenograft, we found CASIN administration (1.2 mg/kg, IV) transiently elicited mobilization of LICs from BM to PB by 20 minutes post injection. This was correlated with in vitro suppression of SDF-1α induced F-actin polymerization detected by FACS analysis and directional migration detected by a transwell assay upon CASIN treatment of the LICs (5-10 μM). Similar observations were made using Cdc42-specific shRNA knockdown of endogenous Cdc42 in the LICs. Continuous CASIN infusion into the xenografted mice for 5 days (1.2 mg/kg, IV, once daily) led to a potent induction of apoptosis of LICs detected by AnnexinV/7AAD staining. Significantly, the CASIN infusion showed no effects on the survival of HCB cells in xenografted SGM3 mice. In addition to a potential niche-dependent survival mechanism, the LICs, not normal HCB cells, appear to directly depend on Cdc42 for survival signals as further in vitro culture studies found that a 24-hour CASIN treatment resulted in a dose-dependent apoptosis of MA9/N-Ras cells, but not of normal HCBs. Finally, mouse genetic studies using MA9 transduced Mx-cre;Cdc42lox/lox BM cells transplanted into congenic BoyJ recipients showed that none of the mice with deleted Cdc42 upon pIpC injection developed AML while all mock-injected mice die from leukemia with less than 4 weeks latency, providing genetic evidence that Cdc42 is required for MA9-induced initiation of AML. Whether CASIN is effective in sensitizing the LICs to conventional chemotherapy in a combinatory regiment is currently under investigation. Our studies present a novel concept that pharmacological targeting of the intracellular signal transducer Cdc42 may have therapeutic value in eradicating LICs.
Disclosures:
No relevant conflicts of interest to declare.
Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis