PKMYT1 Kinase Is an Actionable Synthetically Lethal Target for Del17p Myeloma

In multiple myeloma (MM), deletion 17p (del17p) is one of the most adverse cytogenetic abnormalities and affects 8% of newly diagnosed patients (NDMM). To identify specific actionable vulnerabilities in del17p MM, we analyzed RNA-sequencing data from CD138+ MM cells from 98 NDMM patients harboring del17p and 94 without del17p or TP53 mutation. We combined this data with 2 additional MM patient datasets (608 pts in-house and CoMMpass) and identified 42 recurrently upregulated genes in del17p patients. To prioritize this list, we mined data from the cancer dependency (DepMap) project and found that, among the highly expressed genes in del17p MM, PKMYT1 kinase had the strongest dependency in del17p MM cell lines. Importantly, high expression of PKMYT1 translated clinically into poor overall survival (HR = 3.1, P < 0.0001). We confirmed that PKMYT1 loss-of-function had a greater impact on del17p MM cell lines compared to wild-type (WT) (mean dependency score: -1.3 vs -0.88, P = 0.01) using in-house genome-wide CRISPR KO data in 16 MM cell lines.

PKMYT1 encodes an evolutionarily conserved protein kinase, also known as Myt1. It inhibits CDK1 activity via phosphorylating Thr14 and Tyr15, causing its sequestration in the cytoplasm and disrupting the CDK1-Cyclin B complexes needed for mitosis entrance. The G2-M checkpoint pathway was indeed enriched in del17p patients.

We next evaluated RP-6306 (lunresertib) a selective inhibitor of PKMYT1 catalytic activity for its potential preferential activity against del17p MM cells. We treated a panel of MM cell lines, including 4 with monoallelic del17p, 3 with a TP53-mutation and 3 double-hit along with 5 cell lines with WT TP53. While PKMYT1 inhibition induced CDK1 hyperactivation by impairing the inhibitory phosphorylation on Thr14 in both WT and del17p cells, it caused a significant time and dose-dependent decrease of cell viability in MM cells harboring del17p and/or TP53-mutation, with a significantly lower IC50 in this subgroup compared to WT. Importantly, p53 KO in WT cells sensitized the cells to PKMYT1 inhibition.

Lunresertib treatment caused the accumulation of non-replicating S-phase cells (exhibiting a DNA content between 2C and 4C but not incorporating the synthetic nucleoside BrdU) in del17p MM cell lines but not in WT, indicating replication stress. Indeed, there was a dose and time-dependent accumulation of γH2AX and the induction of replication stress markers (phospho-CHEK2 and phospho-RPA32) in del17p MM cell lines but not in WT cells after PKMYT1 inhibition. Moreover, inhibition increased the number of micronuclei in a dose-dependent manner only in del17p MM cell lines. These features were also observed after treatment of MM cell lines cocultured with bone marrow stromal cells. The addition of a CDK1 inhibitor (RO-3306) partially restored cell viability and abolished lunresertib-induced replication stress, validating that a hyperactivated CDK1 was in part responsible for these effects.

The premature mitotic entry of actively replicating cells is associated with chromosome pulverization (a marker of mitotic catastrophe). As such, we confirmed that PKMYT1 inhibition led to the formation of supernumerary centrosomes and a disorganized spindle and induced chromosome pulverization in del17p cells but not WT. Importantly, PKMYT1 expression was positively correlated with two markers of genomic instability: genomic scar score and mutational load in MM patients, as assessed by WGS data from clinically annotated MM patient cells. We also assessed whether DNA-damaging agents (melphalan, bendamustine) rendered cells sensitive to PKMYT1 inhibition or synergized with it. Indeed, in del17p and double-hit MM cell lines, lunresertib rendered cells sensitive to DNA-damaging agents. Moreover, in WT MM cells, the killing effect of DNA-damaging agent combined with lunresertib was higher than the one of DNA-damaging agent alone.

Finally, PKMYT1 inhibition was very effective in a dose-dependent manner against del17p MM cells in vivo. Of note, lunresertib in phase I/II clinical trials in advanced cancers is well tolerated.

In conclusion, PKMYT1 is an actionable target for MM cells harboring del17p. PKMYT1 inhibition with lunresertib specifically induced DNA damage, mitotic catastrophe, and subsequent cell death in MM cell lines with del17p or TP53 impairment, representing a potential tailored therapeutic option for this high-risk group of MM patients.