We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

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.

The Johnstone laboratory receives research support from Roche, BMS, Astra-Zeneca and MecRx. RWJ is a scientific consultant and shareholder in MecRx. The McArthur laboratory receives non-financial support from Pfizer Oncology for supply of palbociclib.Research.

peptide receptor radionuclide therapy (pRRt) is an important treatment option for patients with somatostatin receptor-2 (SSTR2)-expressing neuroendocrine tumour (NET) though tumour regression occurs in only a minority of patients. Therefore, novel PRRT regimens with improved therapeutic activity are needed. Radiation induced DNA damage repair is an attractive therapeutic target to increase PRRT efficacy and consequently, we have characterised a panel of preclinical models for their SSTR2 expression, in vivo growth properties and response to 177 Lu-DotA-octreotate (Lutate) pRRt to identify models with features suitable for evaluating novel therapeutic combinations. In vitro studies using the SSTR2 expressing AR42J model demonstrate that the combination of LuTate and the small molecule Poly(ADP-ribose) polymerase-1 (PARP) inhibitor, talazoparib led to increased DNA double strand breaks, as assessed by γ-H2AX foci formation, as compared to LuTate alone. Furthermore, using the AR42J tumour model in vivo we demonstrate that the combination of LuTate and talazoparib significantly improved the anti-tumour efficacy of LuTate alone. These findings support the clinical evaluation of the combination of LuTate and PARP inhibition in SSTR2-expressing NET. Neuroendocrine tumours (NET) represent a rare and heterogeneous group of tumours. Characteristically slow growing, these tumours are often diagnosed late in the disease course with locally-advanced or metastatic tumours. A subgroup has associated hypersecretion of hormones that can cause significant morbidity. NET arises from neuroendocrine cells of the diffuse endocrine system with the highest incidence observed in the gastrointestinal tract (70%) and lung (30%) 1,2. Classification of NET is based on the cell-type, organ of origin or associated hormone secretion, while grading is based on the cell differentiation and, in particular, on proliferation rate 3. Clinical management is highly complex and may involve surgery, chemotherapy and molecularly targeted therapies. More recently peptide receptor radionuclide therapy (PRRT) has become well established as a key therapeutic modality for NET, particularly those involving the gastrointestinal tract 4. Neuroendocrine cells are regulated by hormones acting via G-protein coupled receptors such as the somatostatin receptor (SSTR2). Since many NET share high level expression of SSTR2, this feature has been exploited for diagnosis, staging and the therapeutic targeting of NET 5. The 111 In-labelled somatostatin analogue, octreotide and the more recently developed 68 Ga-labelled analogues have been used widely for diagnostic imaging of SSTR2-expressing tumours by SPECT and PET, respectively 6,7. Incorporation of other therapeutic radionuclides such as 177 Lu and 90 Y, optimisation of the chelating agent and replacement of octreotide with the higher affinity

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.