Small Molecule SHIP Agonist AQX-MN100 Inhibits Multiple Myeloma (MM) Cell Growth In NOD/SCID Mice and Reduces Immune Suppressive Myeloid Derived Suppressor Cells (MDSC) and Regulatory T Cells (Tregs) In Mouse Model

Abstract 792

We recently described a novel anti-MM drug (AQX-MN100) which is a small molecule agonist of SHIP (Src homology-2 (SH2) containing inositol-5¢-phosphatase) a signaling molecule found only in hemopoietic cells.(Ong et al, Blood; 110:1942, 2007) The molecule was developed using a high-throughput SHIP enzyme assay to screen an invertebrate marine natural product library and isolate the Pelorol.(Yang et al Org Lett; 7:1073, 2005) SHIP normally functions to negatively regulate the PI3K pathway important to normal hemopoietic cells growth and function. Inappropriate activation of the phosphoinositide 3- kinase (PI3K) pathway has been shown to be involved in the pathogenesis of MM and tumour aggressiveness correlates with the degree of activation. The critical role the PI3K/Akt signaling pathway plays in regulating MM cell survival, has stimulated efforts in designing therapeutics that target this pathway. Pan PI3K inhibitors have limited utility in a clinical setting because of their inhibitory effects on all isoforms of the PI3K family as well as non-PI3K targets. SHIP is an exceptionally good target for MM and other hematopoietic disorders that display elevated PI3K/Akt signaling because its expression is restricted to hemopoietic cells. We have shown that an analogue of Pelorol, AQX-MN100 is able to inhibit PI3K signaling and prevent phosphorylation of Akt. AQX-MN100 induced MM cell line apoptosis mediated by caspase and was specific for SHIP expressing cells which are exclusively hematopoietic. AQX-MN100 also enhances the growth inhibition effects of current myeloma drugs Dexamethasone and Bortezomib on human MM tumour cell lines in vitro. (Kennah et al Expt Hematol; 37:1274, 2009)

In this study we have extended these finding to further evaluate the role of this compound in the treatment of myeloma. NOD-SCID mice were injected in the lateral flanks with 2 million luciferase tagged MM1.S multiple myeloma cells in Matrigel. Tumors were allowed to establish for two weeks and then either AQX-MN100 or vehicle was administered in an oil deposit subcutaneously in the lower flank at a dose of 50 mg/kg every three days. Tumor volume was quantified by imaging on a Xenogen IVIS 200 after 6 and 11 days. These studies demonstrate a significant reduction of tumor volume at 6 days p<0.05 and a highly significant reduction at 11 days p<0.01 in the mice receiving AQX-MN100 as compared to vehicle.

We have shown that AQX-MN100 can directly kill MM cells in in vitro and in vivo. However, based on the known functions of SHIP, we predict that SHIP agonists will additionally target critical steps in MM pathogenesis in vivo, including the ability of MM cells to interact with stromal elements and to subvert the immune system. In order to evaluate this later feature we evaluated the ability of SHIP agonists to reverse the tumor associated immune suppression in MM patients. Tumor and host cell/tumor microenvironment secreted factors promote the production and activation of cells associated with cancer progression: the immune suppressive myeloid derived suppressor cells (MDSC) and regulatory T cells (Tregs). These cells normally regulate immune responses by inhibiting the activation of immune effector cells. The involvement of SHIP in the regulation of these cells is predicted by the observation that MDSC and Treg numbers are elevated in SHIP deficient mice. In this study Balb/C mice, 6 mice/group in duplicate were given either AQX-MN100 3 mg/kg and 10 mg/kg or vehicle once daily orally. At the end of three weeks mesenteric lymph nodes were harvested and subjected to FACS analysis to determine the proportion of MDSC (CD11b+Gr1+) and Treg (CD4+CD25+FoxP3+) cells. Spleen cells were also analysed for B cells, NK cells and granulocytes. In both of the AQX-MN100 treated groups the numbers of MDSC and Tregs were significantly lower than controls while Total CD11b, Total CD3, and spleen B, NK and granulocytes were not different from vehicle treated controls.

The known role of SHIP in regulating hemopoietic cell function and the role of SHIP agonists in MM cell killing as well as additional actions on other aspects of MM pathophysiology may make them a powerful treatment option for MM, either alone or in synergy with other known MM therapies. Further development of this agent for the treatment of MM is ongoing.