Targeting Loss of the Tumor Suppressor TENT5C in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which multiple genomic events lead to tumor development and progression. Loss of TENT5C through mutation or deletion on chromosome 1p occurs in more than 20% of newly diagnosed MM patients and is associated with a poor progression free and overall survival. Although, many studies have identified TENT5C as a tumor suppressor gene in MM, the impact of gene loss on MM physiopathology remain to be elucidated. Here we sought to identify drugs which may specifically target TENT5C-null MM cells using targeted and high-throughput drug screens (HTS).

Methods: We generated CRISPR/Cas9 knockout (ko) of TENT5C in wild type (wt) U266 and KMS-11 MM cell lines and over-expressed (oe) TENT5C in MM cell lines with biallelic inactivation (H929 ^-/- and LP-1 ^-/-). Modified cell lines were compared to their original counterparts for gene expression, proliferation, and cell cycle differences. Targeted and HTS were performed to identify synthetic lethal pathways related to loss of TENT5C in MM that will result in specific cell death.

Results: In TENT5C^ko cells the RNA poly(A) polymerase activity was decreased at two TENT5C top targeted substrates, immunoglobulin kappa light chain and signal sequence receptor. Consistent with TENT5C tumor suppressor function, cell proliferation analysis showed that TENT5C^ko cells had a higher growth rate than TENT5C^wt cells. Cell cycle analysis, after nocodazole synchronization, showed TENT5C-null cells had an increased G2/M population compared to TENT5C-positive cells (U266 TENT5C^ko 19.86% vs. TENT5C^wt 2.6%, p<0.001; H929 TENT5C^-/- 8.22% vs. TENT5C ^oe 1.18%, p<0.001), indicating an increase in cell cycle progression in TENT5C-null cells.

Given our prior data indicating an increase in splicing abnormalities and non-homologous end joining (NHEJ) activation in TENT5C^-/- patient samples, we performed growth inhibition curves using appropriate drugs. The spliceosome inhibitors pladienolide B and isoginkgetin affected TENT5C^ko cells at lower concentrations than TENT5C^wt cells (U266 TENT5C^ko GI ₅₀=0.58 vs. TENT5C^wt GI ₅₀=1.43 nM, U266 TENT5C^ko GI ₅₀=7.03 vs. TENT5C^wt GI ₅₀=13.72 µM, p<0.001, respectively) as did NHEJ inhibitors STL127705 and SCR7 (U266 TENT5C^ko GI ₅₀=22.5 vs. TENT5C^wt GI ₅₀=62.55 µM, U266 TENT5C^ko GI ₅₀=41.93 vs. TENT5C^wt GI ₅₀=117.38 µM, p<0.01). These results were confirmed on the other modified cells to ensure TENT5C specificity.

A HTS using the LOPAC1280 library also identified several hits specifically affecting TENT5C^null cells. The hits were categorized according to their targets to identify pathway enrichment. Inhibitors targeting the cell cycle (roscovitine, apigenin, SP60012) and angiogenesis (amsacrine) were selected for validation and further investigation. The differential effect observed in the HTS data was confirmed with full dose kill-curves on the same cell line (U266) and validated in another TENT5C^ko cell line (KMS-11) to ensure target specificity. For example, amsacrine, an angiogenesis inhibitor, exhibited differential activity onT ENT5C^ko and TENT5C^wt cells (U266 TENT5C^ko GI ₅₀=5.63 µM vs. U266 TENT5C^wt GI ₅₀=9.19, p<0.001; KMS-11 TENT5C^ko GI ₅₀=9.26 µM vs. KMS-11 TENT5C^wt GI ₅₀=14.38, p<0.001). Similarly, roscovitine, a CDK inhibitor, affected TENT5C^ko cells at lower concentrations than TENT5C^wt cells (U266 TENT5C^ko GI ₅₀=23.33 vs. U266 TENT5C^wt GI ₅₀=34.91 µM; KMS-11 TENT5C^ko GI ₅₀=13.47 vs. KMS-11 TENT5C^wt GI ₅₀=29.33 µM, p<0.001). Additionally, cell cycle analysis showed that roscovitine induced a G ₀/G ₁ arrest in a dose-response manner in U266 and KMS-11 cells. Interestingly, TENT5C^ko cells were more sensitive to the CDK inhibitor, and the arrested KMS-11 G ₀/G ₁ populations increased from 3.35 in TENT5C^wt to 6.46 % in TENT5C^ko and from 15.51 in TENT5C^wt to 21.15 % in TENT5C^ko (p<0.05), after treatment with 12.5 and 25 µM of roscovitine, respectively. The same trend was also validated in U266 TENT5C^ko cells to ensure target specificity.

Conclusion: Our results indicate a tumor suppressor role of TENT5C in MM, with antiproliferative properties and a susceptibility to spliceosome and NHEJ inhibitors. The HTP screen identified other targets that are currently undergoing validation leading to the identification of the molecular mechanisms underlying this function.