PIKfyve Inhibitors for the Treatment of Multiple Myeloma

Introduction

Using a high throughput screening approach employing ~80 FDA approved, late stage clinical trial, or drugs of interest to multiple myeloma (MM), we examined single agent drug sensitivity of 35 MM and lymphoma cell lines and in 65 primary MM samples. We identified APY-0201, a PIKfyve inhibitor (P5I), as lethal at nanomolar concentrations in 48% of human myeloma cell lines (HMCLs) and 28.6% of primary MM samples after 24 hour of drug exposure. In 20% of patient samples, in vitro sensitivity was less than 100nM EC50. PIKfyve is a lipid and protein kinase that controls pleiotropic cellular functions, notably regulation of endomembrane homeostasis; mechanisms of sensitivity and resistance were explored.

Methods

Apilimod, a key P5I initially developed as an interleukin (IL)-12 and IL-23 inhibitor, has been used as an immunomodulatory agent in the treatment of inflammatory diseases and has been recently suggested for the treatment of neoplastic diseases, including B-cell Non-Hodgkin's Lymphoma (NHL). YM201636 has also been described as a potent P5I. The cellular efficacies of APY-0201, apilimod, and YM201636 in HMCLs were assessed using the CellTiter Glo assay (Promega), in 20-point 2-fold dilutions of drug concentrations at 72 hours; and HMCL P5I sensitivity was correlated with RNA-SEQ data publically available at www.keatslab.org. Cell lysates were extracted for western blot expression analysis of transcription factor EB (TFEB), cathepsin D, caspase 3, poly (ADP-ribose) polymerase (PARP), at different drug exposure time points (3, 6, 12, 24, 48, and 72 hours). Ex vivo sensitivity to APY-0201, the most potent studied P5I, was measured in CD138+ primary cells of 56 primary MM patients at 24 hours. In vivo activity of apilimod at 100-150 mg/kg and APY-0201 at 30-45 mg/kg was evaluated over 2 weeks in the Vk*MYC transgenic mouse model of MM. Tumor burden was measured by weekly evaluation of M-spike serum levels.

Results

APY-0201 was the most potent P5I, followed by YM201636 and apilimod. However, dose-dependent inhibition of cell viability was observed in all HMCLs. APY-0201 EC50 ranged from 2.8 to 266.7nM (mean 123.4nM), 63.8 to 8425.8nM (mean 1329nM) for YM201636, and 62.5 to 3561.2nM (mean 2026nM) for apilimod. APY-0201 maximum inhibition ranged from 68.2 to 100% (mean 87.2%), 33.2 to 100% (mean 64.6%) for YM201636, and 56.7 to 100% (mean 83.6%) for apilimod. A significant correlation between low RNA-SEQ expression of TFEB, which regulates lysosomal biogenesis and autophagy, and lower sensitivity for APY-0201 was found (Wilcoxon signed-rank test p-value = 0.017). No correlation with baseline OSTM1, CLCN7 or SNX10 levels was seen.

APY-0201 dose dependent sensitivities were observed in 37.5% of ex vivo patient samples. Of the 21 patients demonstrating sensitivity, 2 were inhibited at an EC50<10nM (median 8.58nM), 10 samples presented EC50 between 10 and 100nM (median 64.67nM), and 9 samples presented EC50>100nM (median 1000.16nM). Thus, 12 (21.4%) primary MM patient samples exhibited lethality with less than 100nM concentration of APY-0201 after 24 hours. P5I activity profile was not obviously correlated with clinical findings, such as stage of disease or genetic subtype, in either cell lines or primary samples.

As demonstrated previously in B-cell NHL, treatment of the most sensitive cell line, KMS26, with apilimod and APY-0201 was associated with activation of TFEB, found in its dephosphorylated state. An accumulation of the inactive precursor procathepsin D was also observed in KMS26 following treatment, demonstrating a decrease in maturation of lysosomal proteases. Furthermore, the cytotoxic activity of both compounds was shown by induction of apoptosis makers, i.e. caspase 3 and PARP. In ongoing studies, in vivo activity was evident in 2 of 6 Vk*MYC transgenic MM mice treated with a P5I; dose finding and longer treatment duration studies are underway.

Conclusions

Inhibition of PIKfyve surprisingly demonstrated promising anti-myeloma activity in vitro, ex vivo and in vivo, thus providing a rationale for further evaluation in the treatment of MM. As reported by others in NHL, a novel mechanism of action involving disruption of endolysosomal function is suggested in PIKfyve mediated cytotoxicity in MM.

Acknowledgment

This work was supported in part by the Multiple Myeloma Research Foundation.