PIK-001 a Novel Small Molecule Inhibitor of Pikfyve for Treatment of Multiple Myeloma (MM)
Authors: Stewart A. Keith , Li Zhihua , Issakova Olga , Sepetov Nikolai , Trudel Suzanne 
Published: November 2021

Abstract
We utilized ex vivo chemo-genomics screening to identify potentially unrecognized targets of plasma cell biology. Medium throughput screening on MM and NHL cell lines was conducted using a panel of known FDA-approved oncology drugs, kinase inhibitors, and epigenetic compounds assembled by the investigators. A refined panel was then selected and sensitivity to the single agents was evaluated in 25 MM and 15 NHL cell lines. One panel drug APY-0201 and related drugs such as apilimod were initially developed as suppressors of production of interleukin (IL)-12 and IL-23 production and entered clinical trials for inflammatory diseases but subsequently were found to be primary inhibitors of PikFyve. We have previously published a dose dependent inhibition of cellular viability in all 25 MM cell lines exposed to the PIKfyve inhibitor APY-0201, with a median EC 50 of 55 nM and a median maximum inhibition of cellular viability of 81%. We next tested 100, ex vivo primary CD138+ selected, patient samples. Dose-dependent sensitivities for APY-0201 were observed in 40% of ex vivo samples at only 24 h, with 47% exhibiting an EC 50 lower than 100 nM. The sensitivity to both APY0201 and apilimod was examined in 15 ex vivo primary patients' samples after a longer 72 h incubation, and more significant anti-MM activity was noted with over 90% of the primary patients' samples showed dose dependent inhibition of cellular viability in response to both drugs. APY-0201 was considered the more potent PIKfyve inhibitor, with 71% of the samples exhibiting sensitivity in the nanomolar range (median EC 50 179 nM). We then devised a novel fused triazolo-pyrimidine chemical matter, PIK-001, which builds on known structures and which is confirmed to also selectively inhibit PIKfyve with high potency. PIK-001 was shown to be an effective IL12/23 inhibitor, non-toxic to normal peripheral blood mononuclear cells and exhibits broad activity in B cell malignancy cell lines (Figure) but exhibits selectivity, in that solid tumors (breast, pancreas, renal, cholangio) are much less sensitive or entirely resistant. As with other Pikfyve inhibitors PIK-001 disrupts lysosomal function and, consequently, autophagic flux in B cell malignancy. Autophagy is critical in plasma cells for sustainable immunoglobulin synthesis and endoplasmic reticulum capacity. This high basal necessity of autophagy in antibody producing plasma cells and MM cells indicate the clinical potential of this novel target. After initial dose finding studies mice were inoculated with transgenic MM Vkappa*myc generated tumor and 7 days later PIK-001 treatment was started. PIK-001 was delivered at 60mg/kg per day by oral gavage 6 days a week. 80% of untreated controls developed tumor and died within the first 4 weeks. Only 1 of 5 treated controls (20%) developed tumor, all were alive when the experiment was stopped and all controls were dead. In a second experiment 8 mice were treated beginning on day 7 post inoculation with PIK-001 at 100mg/kg daily, 6 days a week for one month. By day 14 of treatment (21 days since inoculation) control mice had developed large monoclonal spikes whereas treated animals had minimal increases. Between day 21 and 28 post inoculation all controls died. In treated animals the first death occurred on day 39. Summary: Our findings demonstrate inhibition of cellular viability with APY0201, in MM and NHL cell lines, and in over 150 primary ex vivo patient samples. PIK001 is a novel chemical entity with demonstrable PIKfyve inhibition and cellular cytotoxicity on the same scale as APY0201. The specificity of PIK-001 was demonstrated against all tested kinases, GPCRs, ion channels, and enzymes. Preliminary murine studies with PIK001 have shown high efficacy in preventing myeloma growth using a transplantable VKappa*myc cell line and daily oral delivery of drug. This pre-clinical data sets the stage for future clinical testing.
Figure 1 Figure 1.

Disclosures
Stewart: PikSci Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Tempus Inc.: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Genomcs England: Membership on an entity's Board of Directors or advisory committees; Skyline diagnostics: Consultancy; Amgen: Honoraria; BMS: Honoraria; GSK: Honoraria; Janssen: Honoraria; Oncopeptides: Honoraria; Sanofi Aventis: Honoraria. Issakova: PikSci Inc.: Current holder of individual stocks in a privately-held company; nanosyn Inc.: Current Employment, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Sepetov: nanosyn Inc.: Current Employment, Current holder of stock options in a privately-held company; PikSci Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties. Trudel: Genentech: Research Funding; Pfizer: Honoraria, Research Funding; Roche: Consultancy; Sanofi: Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding.