We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes, , and were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.

Microtubules are dynamic structures that must elongate, disassemble, and be cleaved into smaller pieces for proper neuronal development and function. The AAA ATPase Spastin severs microtubules along their lengths and is thought to regulate the balance between long, stable filaments and shorter fragments that seed extension or are transported. In both Drosophila and humans, loss of Spastin function results in reduction of synaptic connections and disabling motor defects. To gain insight into how spastin is regulated, we screened the Drosophila melanogaster genome for deletions that modify a spastin overexpression phenotype, eye size reduction. One suppressor region deleted p21-activated kinase 3 (pak3), which encodes a member of the Pak family of actin-regulatory enzymes, but whose in vivo function is unknown. We show that pak3 mutants have only mild synaptic defects at the larval neuromuscular junction, but exhibit a potent genetic interaction with spastin mutations. Aberrant bouton morphology, microtubule distribution, and synaptic transmission caused by spastin loss of function are all restored to wild type when pak3 is simultaneously reduced. Neuronal overexpression of pak3 induces actin-rich thin projections, suggesting that it functions in vivo to promote filopodia during presynaptic terminal arborization. pak3 therefore regulates synapse development in vivo, and when mutated, suppresses the synaptic defects that result from spastin loss.

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol (PEG) nanoparticles, and antibody-drug conjugates (ADCs). Here, we report the molecular pharmacology of exatecan compared to the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ATR inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533 and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR)-deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.

We identified the PIKFYVE inhibitor apilimod as a potent and selective cytotoxic agent against B-cell nonHodgkin lymphoma (B-NHL). Our data robustly establish PIKFYVE as the target through which apilimod kills B-NHL cells and show that apilimod-induced death in B-NHL is mediated by broad disruption of lysosome homeostasis characterized by lysosomal swelling, TFEB nuclear translocation, impaired maturation of lysosomal enzymes and incomplete autophagosome clearance. Furthermore, through genome-wide CRISPR knockout screening, we identified specific lysosomal genes (TFEB, CLCN7, OSTM1 and SNX10) as critical determinants of apilimod-induced cytotoxicity. Together these data highlight disruption of lysosome homeostasis through PIKFYVE inhibition as a novel anticancer mechanism in B-NHL and potentially other cancers. In our recent manuscript we demonstrated that apilimod, a drug originally brought into the clinic for inflammatory diseases, has highly potent activity against all subtypes of B-cell non-Hodgkin lymphoma in vitro. We also observed antitumor activity in in vivo models of B-NHL as a single agent and in combination with the targeted therapies rituximab and anti-CD274/PD-L1 at drug concentrations achievable in humans. As a result, we recently initiated a multicenter trial (NCT02594384) to test apilimod's efficacy in B-NHL patients.The sensitivity of B-NHL cells to apilimod raises important questions about underlying mechanisms. Apilimod was previously shown to inhibit PIKFYVE (phosphoinositide kinase, FYVE-type zinc finger containing). We found that the activity of apilimod toward PIKFYVE is exquisitely selective, as no offtarget activity was detected across a panel of 456 normal and disease-related protein and lipid kinases. We furthermore isolated an apilimod-resistant mutant of PIKFYVE that renders cells insensitive to this drug.PIKFYVE is an endosomal lipid kinase that is targeted to endosomes via interactions between its FYVE domain and phosphatidylinositol-3-phosphate (PtdIns3P). At endosomes, PIK-FYVE phosphorylates PtdIns3P to generate PtdIns(3,5)P 2 . PtdIns(3,5)P 2 is a defining component of late endosomes and lysosomes and regulates multiples aspects of their function such that PtdIns(3,5)P 2 depletion following PIKFYVE inhibition produces a striking swelling of endolysosomal organelles and induces dephosphorylation and translocation of TFEB, the master regulator of lysosomal biogenesis, into the nucleus. These changes coincide with an increase in LysoTracker staining, and an increase in lysosomal gene expression. Whereas these observations suggest that apilimod-treated cells trigger a homeostatic response that is meant to restore endolysosomal function, we also observed an accumulation of unprocessed cathepsins, and increases in SQSTM1/p62 and LC3-II. The buildup of immature cathespins suggests that this homeostatic response is not successful. Indeed, elevated levels of SQSTM1/p62 and LC3-II) indicates a deficit in the ability of lysosomes from apilimodtreated cells to clear autophagic car...

Key Points The heat shock protein 90 inhibitor LAM-003 displays potent in vitro and in vivo activity as a single agent and combined with venetoclax. LAM-003 retains antileukemic activity against AML cells rendered resistant to FLT3 kinase inhibitors by mutation or stromal signaling.