Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Biosciences

Major Professor

Florian A. Karreth, Ph.D.

Committee Member

Keiran S.M. Smalley, Ph.D.

Committee Member

Elsa R. Flores, Ph.D.

Committee Member

Gina M. DeNicola, Ph.D.

Committee Member

Karen Mann, Ph.D.

Keywords

PHGDH, Genetically Engineered Mouse Models, BRAFV600E, ATF4, Serine Metabolism

Abstract

Metabolic alterations play a critical role in shaping tumor growth and development. Many metabolic enzymes are shown to play an important role in different aspects of tumor development, growth, and metastasis. One such enzyme that we are investigating in this project is the Phosphoglycerate Dehydrogenase (PHGDH). The PHGDH gene, encoding the rate-limiting enzyme of the serine synthesis pathway, is located at chromosome locus 1p12 and frequently amplified in melanoma. PHGDH is required for the proliferation of melanoma cells with chromosome 1p12 amplification. However, the role of PHGDH in chromosome 1p12 non-amplified melanoma is poorly understood. In this project, we sought to investigate the role and regulation of PHGDH in melanomagenesis.

We observed that PHGDH is overexpressed in melanoma cell lines that do not harbor chromosome 1p12 amplification compared to melanocytes. Additionally, PHGDH is upregulated in melanomas compared to nevi. Hyperactivation of the MAPK pathway through activating mutations in BRAF or NRAS occurs in nearly all melanomas. Since PHGDH is universally upregulated in melanoma cells, we hypothesized that oncogenic BRAF mutations upregulate PHGDH expression, thereby generating a metabolic vulnerability in melanoma. Pharmacologic inactivation of the MAPK pathway using inhibitors of mutant BRAF/MEK/ERK resulted in the downregulation of PHGDH expression both in vitro and in allograft tumors in vivo. On the other hand, acute expression of oncogenic BRAF (BRAFV600E) in melanocytes resulted in upregulation of PHGDH RNA and protein expression. Downregulation of PHGDH in 1p12 non-amplified melanoma cell lines decreased their cell proliferation in vitro.

To investigate the role of PHGDH in melanoma formation, we generated a melanoma mouse model containing a genetic knockdown of Phgdh in a BrafV600E/WT; Pten- /- (BPP) background which significantly increased survival as compared to the control. Importantly, PHGDH expression was critical for melanomagenesis as depletion of Phgdh in another genetic mouse model blocked melanoma formation. In addition to de-novo synthesis, serine can be taken up from extracellular compartments. Depletion of extracellular serine from the media resulted in upregulation of PHGDH expression and reduced proliferation of melanoma cells in vitro. By contrast, serine-glycine starvation in BPP mice did not affect tumor development or survival. These results implicate the dependency of melanoma tumors on de-novo serine synthesis through PHGDH. To investigate the therapeutic applicability of the PHGDH dependency, we inhibited the MAPK pathway with BRAF inhibitor along with extracellular serine-glycine depletion. We hypothesized that inhibiting the BRAF-mediated PHGDH expression along with extracellular serine-glycine starvation effectively depletes serine specifically in melanoma cells. Notably, there was a significant increase in cell death when mutant BRAF inhibition was combined with serine-glycine depletion. The cell death by mutant BRAF inhibition alone or in combination with serine-glycine depletion was due to apoptosis, which was rescued by inhibition of the apoptotic pathway. Moreover, our preliminary studies suggest that serine-glycine depletion potentiates the effect of mutant BRAF inhibition on melanoma growth in BPP mouse models. Mutant BRAF inhibition significantly decreased serine levels in the tumors and it did not reduce further as compared to the serine depletion alone. Overall, these results suggest the importance of PHGDH for melanoma cell proliferation and tumor growth irrespective of its amplification status. Combined MAPK inhibition and serine starvation may therefore exploit a metabolic vulnerability of melanoma cells and could be explored for melanoma therapy.

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