By reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry, MGSA-alpha, -beta, -gamma, and CXCR2 mRNA expression and proteins are detected in 7 out of 10 human melanoma lesions. The biological consequence of constitutive expression of the MGSA/GRO chemokine in immortalized melanocytes was tested in SCID and nude mouse models. Continuous expression of MGSA/GRO-alpha, -beta, or -gamma in immortalized melan-a mouse melanocytes results in nearly 100% tumor formation for each of the clones tested, whereas clones expressing only the neomycin resistance vector form tumors <10% of the time. Moreover, antibodies to the MGSA/GRO proteins slow or inhibit the formation of tumors in the SCID mouse model and block the angiogenic response to conditioned medium from the tumor-producing clones. Transcription of the MGSA/GRO chemokines is regulated by an enhancesome-like complex comprised of the nuclear factor-kappaB (NF-kappaB), HMG(I)Y, IUR, and Sp1 elements. In Hs294T melanoma cells the half life of the IKB protein is shortened in comparison to normal retinal epithelial cells, facilitating the endogenous nuclear localization of NF-kappaB. We propose that this endogenous nuclear NF-kappaB, working in concert with the 115-kDa IUR-binding factor, promotes constitutive expression of MGSA/GRO genes.

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Intrinsic resistance of unknown mechanism impedes the clinical utility of inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) in malignancies other than breast cancer. Here, we used melanoma patient-derived xenografts (PDXs) to study the mechanisms for CDK4/6i resistance in preclinical settings. We observed that melanoma PDXs resistant to CDK4/6i frequently displayed activation of the phosphatidylinositol 3-kinase (PI3K)–AKT pathway, and inhibition of this pathway improved CDK4/6i response in a p21-dependent manner. We showed that a target of p21, CDK2, was necessary for proliferation in CDK4/6i-treated cells. Upon treatment with CDK4/6i, melanoma cells up-regulated cyclin D1, which sequestered p21 and another CDK inhibitor, p27, leaving a shortage of p21 and p27 available to bind and inhibit CDK2. Therefore, we tested whether induction of p21 in resistant melanoma cells would render them responsive to CDK4/6i. Because p21 is transcriptionally driven by p53, we coadministered CDK4/6i with a murine double minute (MDM2) antagonist to stabilize p53, allowing p21 accumulation. This resulted in improved antitumor activity in PDXs and in murine melanoma. Furthermore, coadministration of CDK4/6 and MDM2 antagonists with standard of care therapy caused tumor regression. Notably, the molecular features associated with response to CDK4/6 and MDM2 inhibitors in PDXs were recapitulated by an ex vivo organotypic slice culture assay, which could potentially be adopted in the clinic for patient stratification. Our findings provide a rationale for cotargeting CDK4/6 and MDM2 in melanoma.