Page 185 - Haematologica - Vol. 105 n. 6 - June 2020

PIKfyve inhibition in multiple myeloma
sensitivity to both APY0201 and apilimod was then exam- ined in 15 ex vivo primary patients’ samples after 72 h incu- bation, and more significant anti-MM activity was noted. Over 90% of the primary patients’ samples showed dose- dependent inhibition of cellular viability in response to both drugs. APY0201 was considered the more potent PIKfyve inhibitor, with 71% of the samples exhibiting sensitivity in the nanomolar range (median EC50 179 nM), while for apilimod, 93% of the samples exhibited sensitiv- ity in the micromolar range (median EC50 22618 nM) (Table 2 and Online Supplementary Table S3).
The cytotoxicity of APY0201 for different leukocyte populations was evaluated in two primary patients’ sam- ples with increasing drug concentrations. The plasma cell population of the first sample was sensitive to APY0201, with a marked decrease in cellular viability when incubat- ed with the highest dose of the drug (Online Supplementary Figure S7A); the second sample was consid- ered resistant (Online Supplementary Figure S7B). We did not observe any significant off-target effect of APY0201 in the T and B lymphocyte populations in either sample, while some off-target effects were noted in the popula- tion composed of monocytes, macrophages, and granulo- cytes.
Ploidy and t(11;14) are biomarkers of sensitivity
In HMCL, APY0201 was active regardless of the pres- ence or absence of IgH translocations, TP53 deletion, CKS1B gain, monosomy 13, and MYC rearrangements (Online Supplementary Figure S8). We then evaluated pos- sible associations between APY0201 sensitivity in pri- mary patients’ samples and clinical findings. We exam- ined different disease stages (monoclonal gammopathy of undetermined significance, smoldering MM, and MM), disease status (newly diagnosed or relapsed), fluorescence in situ hybridization cytogenetics [t(11;14), t(4;14), t(14;16), t(6;14), deletion of 17p, CSK1B duplication, MYC rearrangement, monosomy 13, and trisomies of one or more odd-numbered chromosomes], flow cytometry analysis (hyperdiploid status and S-phase), and hyper- diploidy according to Wuilleme et al.25 Despite the limited sample size, primary patients’ samples lacking trisomies (Fisher test, P=0.0232) and harboring t(11;14) (Fisher test, P=0.0189) were more frequently classified as inactive to APY0201 in the ex vivo drug screen (Figure 1A, B). With high-risk patients defined as those with t(4;14), t(14;16), deletion of 17p, or CSK1B duplication, no discernible dif- ference in PIKfyve sensitivity was observed between patients with high- or low-risk genetics.
Data publicly reported from the CoMMpass project (these data were generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives; https://research.themmrf.org and www.themmrf.org) showed lower PIKFYVE expression levels in hyperdiploid samples when compared to non-hyperdiploid samples (t- test, P=0.0003) and in samples lacking t(11;14) when com- pared to samples harboring the translocation (t-test, P<0.0001). These findings were also confirmed in a cohort of 487 primary MM samples (Mann-Whitney test, P<0.0001) (unpublished data). We hypothesize that increased PIKFYVE expression levels may be associated with increased resistance to PIKfyve inhibitors, with high- er concentrations of the inhibitors necessary to activate TFEB. However, further studies are needed to support this hypothesis.
Upregulation of lysosomal pathways in vitro and ex vivo
To further elucidate the anti-MM mechanism of PIKfyve inhibitors, we evaluated the mRNA-sequencing profiles of five HMCL with low, intermediate and high sensitivities to APY0201. When comparing RNA expres- sion profiles from each HMCL treated with APY0201 for 24 h with its untreated control using the Enrichr platform, gene targets of the lysosomal pathway were upregulated in all five HMCL (P≤0.01), corroborating previous find- ings.26 This is consistent with previous data from B-NHL suggesting that apilimod impaired lysosomal function and consequent degradation of the autophagosomal cargo.12
Three primary patients’ samples identified as sensitive to APY0201 (EC50 of 56.29, 101.14, and 103.36) and one resistant sample, lacking a dose-response curve (Online Supplementary Figure S9), were sent for mRNA-sequencing analysis. The three sensitive patients’ samples harbored
Table 1. Cellular viability inhibition of 20 human myeloma cell lines after 72 h of incubation with three PIKfyve inhibitors.
Mid-point EC50
APY0201
Samples %
HMCL
YM201636 Apilimod
Samples % Samples %
<100nM 5/20 25 0/20 0 0/20 0
100-1000nM 8/20 40 8/20 40 1/20 5
>1000nM 7/20 35 12/20 60 19/20 95
EC50: half maximal effective conentration; HMCL: human myeloma cell lines.
Table 2. Cellular viability inhibition of ex vivo primary patients’ samples after 24 and 72 h incubation with APY0201 and apilimod.
Mid-point EC50
APY0201
24 Hour Incubation Samples %
Ex vivo Primary Patients’ Samples APY0201
72 Hour Incubation Samples %
Apilimod
72 Hour Incubation Samples %
<10nM 2/100 2
3/14 21.4 0/15 0.0
2/14 14.3 0/15 0.0 5/14 35.7 1/15 6.7 3/14 21.4 14/15 93.3 1/14 7.2 0/15 0.0
10-100 nM 100-1000 nM >1000 nM Inactive
17/100 17.0 11/100 11.0 10/100 10.0 60/100 60.0
EC50: half maximal effective conentration.
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