Graduation Date

Spring 5-9-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Molecular Genetics & Cell Biology

First Advisor

Kyle J. Hewitt

Abstract

Sterile alpha motif-containing protein 14 (Samd14) is upregulated in mouse models of anemia and its absence leads to lethality after severe hemolytic anemia, without affecting steady-state hematopoiesis. Samd14 is known to promote SCF/Kit signaling and erythroid colony-forming activity through its sterile alpha motif (SAM), but the precise mechanism remained elusive. The goal of my dissertation was to identify the molecular interactor for Samd14-SAM and establish how the interaction drives erythroid progenitor cell (EPC) expansion during anemia recovery.

To elucidate how Samd14 helps mice recover from acute stress, we conducted a global phosphoproteomic screen in Lineage-Kit+ cells from phenylhydrazine treated anemic mice. We discovered that SCF/Kit signaling inhibits autophagy in wild-type mouse splenic EPCs and this response was attenuated in Samd14-deficient EPCs. Moreover, autophagy gene signatures were deregulated in mouse models of acute (phenylhydrazine-induced) and chronic (Sickle Cell Disease) anemia.

Samd14 interacted via its SAM domain with phosphatidylinositol 3-phosphate (PI3P), which is an integral component of endosomal and autophagic membranes. In protein-lipid overlay assays, PI3P-binding was lost after mutation of a polybasic motif (PBM) within Samd14’s SAM domain. Additionally, proximity ligation assays (PLA) showed decreased PLA signal in SAM-deleted mutant and PBM mutant compared to full length Samd14. Fewer burst-forming erythroid colonies (BFU-Es) were observed in EPCs expressing the SAM-deleted and PBM-mutant indicating that Samd14-PI3P interaction promotes erythroid colony formation.

PI3P’s role in erythroid differentiation was tested using an inhibitor of Class III PI3-kinase VPS34, which is a major source of PI3P in cells. VPS34 inhibition blocked erythroid maturation across four distinct mouse/human erythroid culture systems. Higher doses of VPS34 inhibition were required to block erythroid differentiation in Samd14-deficient cells, suggesting that Samd14 sensitizes cells to VPS34 inhibition. Importantly, VPS34 inhibition suppressed autophagic flux in EPCs but Samd14-deficient cells showed increased flux within immature erythroid-committed populations. We propose a model where Samd14-PI3P molecular association serves to balance autophagic flux, preventing premature differentiation during expansion of stress erythroid progenitors. Collectively, my research provides a mechanistic framework for how Samd14-PI3P axis coordinates autophagy to ensure an effective regenerative response during anemia recovery.

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