Overcoming intrinsic and acquired drug resistance of melanoma is a major challenge. Melanomas are relatively unique in cancer biology in being intrinsically resistant to nearly every chemo-therapeutic agent tried thus far 
. Novel therapies targeting the BRAF oncogene, which is mutated in almost 50% of all melanomas, have demonstrated clinical benefit in melanoma patients. However, repeated experience has shown that melanomas acquired resistance even to new agents selectively targeting BRAF 
. In this study, we characterize an inhibitor of CDKs with remarkable pro-apoptotic activity against human melanoma cells grown under 2D adherent culture conditions, 3D organotypic culture conditions, and in mouse xenografts.
There is currently much interest in the idea of “personalized cancer therapy”, a paradigm whereby patients are selected based on the genetic/mutational profiles of their tumors and given targeted therapies directed against the driving oncogenic mutations. This approach has shown success in melanoma, where carefully selected patients harboring BRAFV600E activating mutations are receiving inhibitors of the MAPK pathway, which is constitutively active due to this mutation 
. This approach is also self-evident when considering CDK’s as a potential melanoma-specific therapeutic target. Previous work has shown that melanoma sub-groups exist with either high or low expression levels of CDK2, with the low CDK2 expressing cell lines posited to be the optimal group for receiving CDK2 inhibitor therapy 
. An initial screen of our panel of melanoma cell lines identified a group of melanoma cell lines with high CDK2 expression and a group with low CDK2 expression. At the RNA level, there was good positive correlation between levels of CDK2 and the transcription factor MITF in both BRAF-V600E
mutated melanoma cell lines. Interestingly, there was also some stratification between CDK2 expression level and BRAF/NRAS
mutational status. We also found that all of the NRAS
mutated melanoma lines evaluated (or included in our panel) had low expression of CDK2, whereas there was some variability within the BRAF
-V600E mutated group, with some cell lines harboring high expression of CDK2 and some with low expression. These results are consistent with CDK2 being a downstream target of the MITF transcription factor in melanoma 
, as previous work has shown that all of the cell lines in the NCI-60 cell line panel with MITF amplification also harbored a BRAF
V600E mutation 
. Although we saw agreement between CDK2 and MITF levels at the RNA level, there was some variability in the levels of MITF protein expression that did not always match with CDK2 protein levels. It is likely that this lack of clear correlation is a consequence of the complex system of post-translational modification and proteasomal targeting seen with MITF.
The observed anti-melanoma effects most likely result from cdk inhibition, though secondary effects of the compound cannot be ruled out.
Treatment of a panel of melanoma cell line with dinaciclib led to a very potent inhibition of cell growth and induction of apoptosis. There was no correlation between CDK2 expression levels and sensitivity of the cell lines to dinaciclib, suggesting that dinaciclib is not functioning exclusively as a CDK2 inhibitor. As it has been previously shown that the BRAF
V600E mutation conveys sensitivity of melanoma cells to BRAF 
and MEK 
inhibitors and the phenothiazine compounds 
, we next addressed whether the presence of a BRAF
mutation conferred sensitivity to dinaciclib. We observed that there was no apparent correlation between dinaciclib response and BRAF/NRAS
mutational status, leading us to conclude that dinaciclib had broad-spectrum anti-melanoma activity and was not conclusively mutation specific.
A number of previous studies have shown that intracellular signaling and drug resistance are modified by the tumor microenvironment. In particular, our group has previously shown that melanoma cells derived from metastases become almost completely resistant to certain inhibitors when grown under 3D organotypic cell culture conditions 
. We found that both the BRAF
-mutated 1205Lu and NRAS
-mutated WM1366 melanoma cell lines retain their sensitivity to dinaciclib when grown as 3D collagen-implanted spheroids, suggesting that modulating the tumor microenvironmental conditions does not increase resistance to this compound. Other melanoma targeted therapies, such as the MEK inhibitors, are associated with reversible cytostatic effects when grown under 3D organotypic cell culture and in vivo
. Here we show that a 14-day dinaciclib treatment of established WM1366 melanoma tumors in vivo
led to a marked inhibition of tumor growth and some degree of regression, suggesting that dinaciclib was having significant cytotoxic effects beyond induction of G2/M phase cell cycle arrest. Biochemically, dinaciclib treatment led to reduced expression of the anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP. Downregulation of these anti-apoptotic proteins is a common feature of other CDK inhibitors, particularly those that have selectivity for CDK9 
. Thus it has been shown that both UCN-01 and flavopiridol decrease the expression of Mcl-1, XIAP, BAG-1, and Bcl-2 in B-cell chronic lymphocytic leukemia cells 
. The downregulation of pro-apoptotic gene expression following CDK9 inhibition is likely to be one of the major pro-apoptotic mechanisms of these compounds. In particular, the ability of dinaciclib to regulate these anti-apoptotic proteins is likely to be of great utility in melanoma where overexpression of XIAP 
, Bcl-2 
and Mcl-1 
is commonplace and can contribute to melanoma chemoresistance.
Dinaciclib treatment also led to the induction of p53 expression under both in vitro
and in vivo
conditions. The role of p53 in melanoma and its potential role in therapy remains somewhat unclear since melanomas generally harbor very low rates of p53 mutations and are generally poor at undergoing p53-dependent apoptosis. Previous work from our group has shown that melanomas can tolerate high levels of p53 expression and do not undergo apoptosis 
. The lack of p53-dependent apoptosis under these conditions is likely a consequence of high expression levels of the E3-ubiquitin ligase MDM2 (or HDM2) 
, which holds the transcriptional activity of p53 in check by targeting it to the proteasome. More than 50% of primary invasive and metastatic melanomas overexpress HDM2 which contributes to the lack of p53 activity in melanoma 
. More recently, it has been shown that MDM4 is upregulated in 65% of melanomas promoting tumor survival by counteracting the effects of p53 
. However, under certain conditions, p53 can become pharmacologically activated in melanoma cells. We and others have previously demonstrated that the pharmacological activation of p53 via either GSK3β inhibition or Nutlin-3 induced high levels of apoptosis in melanoma cells 
. Activation of p53 also seems to be a critical determinant of dinaciclib-induced apoptosis, with selective knockdown of p53 expression leading to a marked inhibition of dinaciclib-induced apoptosis. Interestingly, knockdown of p53 expression was still associated with the dinaciclib-induced reduction in the expression of Mcl-1. This demonstrates either that loss of Mcl-1 was not critical to the pro-apoptotic activity of dinaciclib or that the reduced expression of Mcl-1 still requires functional p53 to induce apoptosis.
Dinaciclib has shown preclinical in vitro and in vivo activity in pancreatic, osteosarcoma and melanoma models 
. Dinaciclib showed moderate activity in a phase 2 trial of melanoma with acceptable toxicity 
. Our work mechanistically supports the anti-melanoma activity of dinaciclib and further demonstrates its potential as a novel therapy for this disease. In addition, the ability of dinaciclib to pharmacologically activate p53 suggests that dinaciclib could be an ideal candidate for combinations with cytotoxic chemotherapies, as a possible mechanism to overcome chemoresistance. This idea fits well with previously published studies showing that flavopiridol potentiates the induction of apoptosis seen following treatment with mitomycin C, paclitaxel, SN-38, and topoisomerase I inhibitors in gastric and breast cancer cell lines 
. In these studies, p53 function was critical for the flavopiridol’s ability to potentiate chemotherapy-induced apoptosis, with the extent of apoptosis induced being 5-fold greater in p53 (+/+) HCT116 cells compared to that seen in p53 (−/−) HCT166 cells 
. Since most melanomas lack p53 mutations 
, it can be reasoned that dinaciclib should have good pro-apoptotic activity across the spectrum of melanoma patients when combined with agents such as those targeted against BRAF/MEK signaling. Data have previously shown that melanoma cells do not undergo apoptosis following MEK inhibitor treatment as a result of constitutively high Mcl-1 expression levels 
. A compound such as dinaciclib, which downregulates Mcl-1 expression, could be an ideal combination candidate in future preclinical and clinical studies.