(PA-299) A35, a Novel Small Molecule Kinase Inhibitor, Shows High Efficacy in Multiple Myeloma and Overcomes Imids Resistance

RAS mutations activate multiple kinase pathways and occur in 50% of multiple myeloma (MM) patients. The mutation frequency increases as the disease progresses and is associated with a poorer prognosis. Furthermore, despite significant advances in immunotherapy, patients eventually develop resistance, highlighting the need for novel treatment strategies, particularly those that target kinase pathways beyond immunotherapies. In an effort to develop small-molecule kinase inhibitors, we developed A35, a novel kinase inhibitor for the treatment of MM.

Methods:   

The anti-MM efficacy of A35 was evaluated on a panel of human MM cell lines using proliferation, apoptosis, and cell cycle assays. The impact on kinase signaling pathways was assessed by western blot assays in MM.1S and LP1 cells following 24 hours of A35 treatment. Kinase activity profiling of A35 was investigated using scanMAX KINOMEscan Lead Hunter Panel (Eurofins). In vivo anti-MM effects were tested using the MM.1S xenograft model in SCID mice, where mice received oral gavage of A35 at doses of 5 mg/kg or 20 mg/kg, or vehicle control for eight weeks.  

Results:   

A35 demonstrated potent anti-proliferative activity across all tested MM cell lines (MM.1S, H929, KMS12-PE, LP1, OPM2, SKMM2, RPMI-8226, JJN3), with an average IC50 of 1.6µM (0.5-3.7µM). Annexin-V/PI staining showed a significant increase of apoptosis in A35 treated cells from 12% in the control to 46% at 2.5µM and 71% (p< 0.05) at 5µM in LP1 and from 20% to respective 32% and 51% (p< 0.05) in MM.1S cells. A35 treatment was associated with a significant G2 cell cycle arrest from 25% to 41% (MM.1S) and 17% to 46% (LP1) (p< 0.05). Western blotting revealed a decrease of p-ERK1/2, p-MEK1/2 and p-AKT without affecting the total protein levels. Notably, c-MYC expression decreased markedly, alongside a modest reduction in BCL2 upon treatment. Kinase inhibition profiling revealed FLT3, HASPIN, JAK1, RSK1 and RSK4 to be the top inhibited kinases with a remaining kinase activity < 20% at 1µM.

Next, we investigated the A35 effects in the setting of IMiDs resistance using RPMI-8226 cells, or the H929 Len^Res cells with acquired lenalidomide resistance. A35, but not lenalidomide, induced significant cell proliferation inhibition (p< 0.05) and c-Myc degradation in both cells, similar to the sensitive parental H929 cells.

In vivo treatment with A35 lead to a prolonged median survival of mice from 29 days (vehicle) to 42 (5mg/kg) and 49 (20mg/kg). There were no significant differences in weight change between the treatment groups and no major side effects.

 

Conclusions:   

A35 is a promising novel multi-kinase inhibitor with robust anti-myeloma activity in vitro and in vivo, including efficacy in IMiD-resistant models. It represents a potential novel therapeutic option for refractory/relapsed myeloma patients, including those who have failed immunotherapy.