Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour characterised by a 7% 5-year survival rate, reflecting early metastasis and rapid resistance to first-line standard-of-care (SoC) platinum/etoposide chemotherapy. The addition of immunotherapy marginally improves survival in a few cases, and alternative approaches are needed. Given the frequent loss-of-function in RB1 and TP53 and aberrant expression of DNA damage repair molecules, targeting cell-cycle-related vulnerabilities is a rational therapeutic strategy for SCLC. Inhibition of protein kinase, membrane-associated tyrosine/threonine 1 (PKMYT1), that regulates M phase entry, has shown promise as an anti-tumour strategy, especially in cyclin E (CCNE1)-amplified tumours due to synthetic lethality between the CCNE1-CDK2 complex and PKMYT1 (Gallo et al. 2021). RP-6306, a PKMYT1 inhibitor, is in phase 1 clinical trials of combination treatments for advanced solid cancers. We asked if PKMYT1 inhibition could be of benefit in SCLC.

SCLC circulating tumour cell (CTC)-derived eXplants (CDX), generated via engraftment of patients’ circulating tumour cells (CTCs) into NSG mice (Hodgkinson et al. 2014), were used as clinically relevant pre-clinical ex vivo and in vivo research platform. Multi-omic profiling (genomic, transcriptomic and proteomic) was used to identify predictive biomarkers of PKMYT1 inhibitor response. RP-6306 efficacy and mechanism were assessed ex vivo using cell viability assays, immunofluorescence, western blotting and flow cytometry. In vivo RP-6306 efficacy was tested in combination with SoC.

Phospho-proteomic profiling of 12 CDX models from chemo-naïve and relapsed patients revealed dysregulated Cyclin-Dependent Kinase 2 (CDK2) phosphorylation activity, frequently more active at disease progression. In both patients with SCLC and CDX models, transcriptomic analysis showed high CCNE1 expression and MMB-FOXM1 signature score along with CDK2 activation, which are known predictors of sensitivity to PKMYT1 inhibition. RP-6306 treatment ex vivo resulted in a range of sensitivities in CDX models (EC50 0.06-10 μM) with CDK2 phosphorylation activity and CDK2 and PKMYT1 mRNA levels predicting response to RP-6306 single agent ex vivo. PKMYT1 target CDK1(T14) was phosphorylated in all 12 CDX models assessed. RP-6306 demonstrated additivity with SoC in the 5 CDX models tested ex vivo. Mechanistically, it induced DNA damage, replication arrest, and cell death in the G2/M cell cycle phase across 3 RP-6306-sensitive CDX models. In vivo, RP-6306 in combination with SoC showed significant benefit over SoC alone in CDX30P.

We showed, in multiple SCLC patient-faithful CDX models, that PKMYT1 inhibition induces DNA damage and apoptosis ex vivo and contributes to tumour growth control in vivo, and identified multiple candidate predictive biomarkers of PKMYT1 inhibition sensitivity.