Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

AbuHammad S ，Cullinane C ，Martin C ，Bacolas Z ，Ward T ，Chen H ，Slater A ，Ardley K ，Kirby L ，Chan KT ，Brajanovski N ，Smith LK ，Rao AD ，Lelliott EJ ，Kleinschmidt M ，Vergara IA ，Papenfuss AT ，Lau P ，Ghosh P ，Haupt S ，Haupt Y ，Sanij E ，Poortinga G ，Pearson RB ，Falk H ，Curtis DJ ，Stupple P ，Devlin M ，Street I ，Davies MA ，McArthur GA ，Sheppard KE ... -  《-》

Palbociclib synergizes with BRAF and MEK inhibitors in treatment naïve melanoma but not after the development of BRAF inhibitor resistance.

Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.

Martin CA ，Cullinane C ，Kirby L ，Abuhammad S ，Lelliott EJ ，Waldeck K ，Young RJ ，Brajanovski N ，Cameron DP ，Walker R ，Sanij E ，Poortinga G ，Hannan RD ，Pearson RB ，Hicks RJ ，McArthur GA ，Sheppard KE ... -  《-》

Functional Determinants of Cell Cycle Plasticity and Sensitivity to CDK4/6 Inhibition.

Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors has emerged as a major obstacle that hinders their utility beyond ER+ breast cancer. In this study, CDK4/6-dependent and -resistant models were employed to identify functional determinants of response to pharmacologic CDK4/6 inhibitors. In all models tested, the activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity. While depleting CDK4 and 6 was sufficient to limit proliferation in specific resistance settings, RB loss rendered cells completely independent of these kinases. The main downstream target in this context was the activation status of CDK2, which was suppressed with CDK4/6 inhibition in an RB-dependent fashion. Protein levels of p27 were associated with plasticity/rigidity of the cell cycle and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via inhibition of SKP2 and targeting the MEK/ERK pathway enhanced the cytostatic effect of CDK4/6 inhibitors. Mice bearing ER+ xenografts displayed a durable antitumor response to palbociclib; however, over the course of treatment, few cells retained RB phosphorylation, which was associated with limited p27 protein levels as determined by multispectral imaging. Similarly, combination treatment of palbociclib with a MEK inhibitor in pancreatic cancer PDX models upregulated p27 and further enhanced the in vivo tumor response to palbociclib. Collectively, these results suggest that the cell cycle plasticity, which enables tumor models to evade palbociclib-mediated activation of RB, could be targeted using a clinically applicable CDK2 inhibitor. SIGNIFICANCE: This work provides a mechanistic insight toward understanding the functional roles of multiple cell cycle regulators that drive plasticity and sensitivity to CDK4/6 inhibition.

Kumarasamy V ，Vail P ，Nambiar R ，Witkiewicz AK ，Knudsen ES ... -  《-》

Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2.

Pack LR ，Daigh LH ，Chung M ，Meyer T ... -  《Nature Communications》

PRMT5 and CDK4/6 inhibition result in distinctive patterns of alternative splicing in melanoma.

Drugs targeting cyclin-dependent kinases 4 and 6 (CDK4/6) are promising new treatments for melanoma and other solid malignancies. In studies on CDK4/6 inhibitor resistance, protein arginine methyltransferase 5 (PRMT5) regulation of alternative splicing was shown to be an important downstream component of the CDK4/6 pathway. However, the full effects of inhibition of CDK4/6 on splicing events in melanoma and the extent to which they are dependent on PRMT5 has not been established. We performed full-length mRNA sequencing on CHL1 and A375 melanoma cell lines treated with the CDK4/6 inhibitor palbociclib and the PRMT5 inhibitor GSK3326595 and analysed data for differential gene expression and differential pre-mRNA splicing induced by these agents. Changes in gene expression and RNA splicing were more extensive under PRMT5 inhibition than under CDK4/6 inhibition. Although PRMT5 inhibition and CDK4/6 inhibition induced common RNA splicing events and gene expression profiles, the majority of events induced by CDK4/6 inhibition were distinct. Our findings indicate CDK4/6 has the ability to regulate alternative splicing in a manner that is distinct from PRMT5 inhibition, resulting in divergent changes in gene expression under each therapy.

Chan LH ，Wang P ，Abuhammad S ，Lim LRJ ，Cursons J ，Sheppard KE ，Goode DL ... -  《-》