Page 190 - Haematologica - Vol. 105 n. 6 - June 2020

C. Bonolo de Campos et al.
Similarly, in primary patients’ samples, an APY0201- mediated increase in autophagic vacuoles was inversely proportional to the EC50 for the PIKfyve inhibitor APY0201 (Figure 5B). Analogous findings were also seen when HMCL and primary patients’ samples were treated for 18 h with apilimod (data not shown). This could, there- fore, be an efficient and fast assay to predict PIKfyve inhibitor response in MM patients.
Discussion
Although the clinical outcome of MM patients with current therapy protocols has improved markedly,35,36 most patients ultimately relapse.37 The identification of novel anti-MM agents therefore remains an important step towards disease control. We first identified the PIKfyve inhibitor APY0201 as a promising anti-MM ther- apeutic in a preliminary screen and included the PIKfyve inhibitor in a 76-drug panel used for more intensive drug screening for sensitivity. Following 24 h incubation of 25 HMCL and 100 ex vivo primary patients’ samples with APY0201, this drug was demonstrated to have activity in >90% and 40%, respectively. PIKfyve inhibition was then validated with three different PIKfyve inhibitors, APY0201, YM201636, and apilimod, generating dose- dependent responses in 20 HMCL, while APY0201 and apilimod showed activity in >90% of ex vivo patients’ samples with a longer 72 h incubation.
The PIKfyve selectivity of apilimod had been demon- strated when this drug was profiled against several kinas- es and no off-target activity was detected,12,38 while the specificity of APY0201 was demonstrated against all test- ed kinases, G-protein-coupled receptors, ion channels, and enzymes, with APY0201 showing superior selectivity over apilimod.14 YM201636 is less selective, inhibiting PIKfyve and insulin-induced activation of class IA PI3 kinase.39 Our findings clearly demonstrated increased inhibition of cellular viability with APY0201, when com- pared to YM201636 and apilimod, in HMCL, NHL cell lines, and primary ex vivo patients’ samples.
PIKfyve negatively regulates TFEB,26 and PIKfyve inhibitor sensitivity has been correlated with higher base- line levels of TFEB, a master regulator of the function of lysosomes and autophagy.29 Following APY0201 treat- ment, TFEB was found in a dephosphorylated state which correlated with its translocation to the nucleus in representative sensitive and resistant HMCL, as previous- ly noted by others.12,40 A PtdIns-3,5-P2-dependent regula- tion of the activation and nuclear translocation of TFEB has also been previously demonstrated.40 In addition, the endolysosomal swelling phenotype associated with loss of PIKfyve function has been linked to a concomitantly reduced number and increased volume of autophagy
organelles after coalescence.26 These findings clearly link PIKfyve inhibitors with lysosomal and autophagic disrup- tion and were confirmed in our analysis.
Impaired lysosomal degradation of autophagic cargo induced by apilimod driving cell death was suggested in B-NHL.38 However, we found an accumulation of lysoso- mal protease precursors, autophagosome and lysosome protein markers, intracellular vacuolization, as well as transcriptomic increases in the lysosome pathway in rep- resentative HMCL both sensitive and resistant to APY0201. This indicated that PIKfyve-induced cell death in MM could not be fully explained by the model of apil- imod’s mechanism of action in B-NHL,38 whereby PIKfyve inhibition led to impaired lysosomal homeostasis with nuclear translocation of TFEB and vacuole forma- tion, causing cell death. We theorize and provide support- ing evidence that one mechanism of resistance to the PIKfyve inhibitor APY0201 in HMCL is by partially main- taining autophagic flux, which decelerates significant imbalances in membrane trafficking and the resulting metabolic alterations.
Autophagy is critical in plasma cell ontogenesis for sus- tainable immunoglobulin synthesis and endoplasmic reticulum capacity, increasing cellular viability, which may be even more important in plasma cell dyscrasias.41 Higher basal protein levels of TFEB were shown in HMCL sensitive to APY0201; therefore, since TFEB over- expression has been associated with increased autophagic flux,42 basal autophagic flux in HMCL could be directly related to the sensitivity to PIKfyve inhibition. MM cells are notably dependent on autophagy for their survival,43,44 thus targeting autophagy via PIKfyve inhibition could represent an effective treatment option for MM.
A phase 1 clinical trial with apilimod in B-cell malignan- cies is in progress, with preliminary results showing promising early antitumor activity in heavily pretreated patients and a favorable safety profile at doses of ≤125 mg BID.45 Ex vivo drug screening is suggested to enrich MM patients sensitive to PIKfyve inhibitors in future clinical trials. Detection of autophagy in response to drug expo- sure is also a promising test to predict sensitivity, although additional validation is necessary. In addition, we anticipate greater sensitivity to PIKfyve inhibitors in patients with trisomies of one or more odd-numbered chromosomes and less sensitivity in MM samples harbor- ing t(11;14).
In summary, we demonstrated promising anti-myelo- ma activity in vitro and ex vivo as a result of inhibition of PIKfyve, a novel therapeutic target in MM. PIKfyve inhibitors disrupt lysosomal function and, consequently, autophagic flux, and the high basal necessity of autophagy in plasma cells and MM cells indicate the clin- ical potential of this novel target in anti-MM strategies. PIKfyve inhibition should be further evaluated in MM.
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