Kashyap Tadisina
The Role of CHOP 10 in UPR-Induced Apoptosis in Severe Congenital Neutropenia Granulocytic Precursors
Mentor:  Dr. Daniel Link, Departments of Internal Medicine and Pathology and Immunology, Washington University School of Medicine

Severe congenital neutropenia (SCN), is a rare heterogenous group of blood disorders whose primary features are: dramatically decreased numbers of peripheral neutrophils and an accumulation of promyelocytes and myelocytes in the bone marrow. This leaves patients extremely susceptible to infections. In addition, these patients have a marked predisposition to develop acute myeloid leukemia. The most common cause of SCN is mutations in the ELA2 gene, which encodes for the enzyme neutrophil elastase (NE) and is highly expressed in the promyelocyte stage of granulocytic differentiation.

However, the molecular mechanisms by which the ELA2 mutations disrupt granulocytic differentiation are unknown. The Link Lab has generated evidence suggesting that the ELA2 mutations lead to the production of misfolded NE protein, activation of the UPR, and ultimately apoptosis of granulocytic precursors.

Previous studies in the lab have established that expression of mutant NE in primary cultured granulocytic precursors leads to UPR-induced apoptosis. The specific hypothesis of my work this summer was to determine if induction of CHOP expression mediated apoptosis. CHOP, a pro apoptotic transcription factor, has previously been shown to be transcriptionally upregulated by endoplasmic reticulum stress.

We first developed a quantative realt time RT-PCR assay for CHOP. As a control, cultured human promyelocytes were treated with tunicamycin (TN), to activate UPR-mediated apoptosis. RNA was prepared from these cells and real time RT_PCR for CHOP and Beta-actin, a housekeeping gene, was performed. Compared with untreated cells, CHOP mRNA relative to Beta-actin mRNA was increased almost 200 fold. Next, we measured CHOP expression in transfected promyelocytes transiently expressing mutant NE, which induces the UPR in a more physiologically relevant manner. However, significant expression of CHOP was not detected. Due to the low numbers of viable cells from which we harvested RNA for this assay, we suspect that this negative result might be partially due to insufficient RNA for our qRT-PCR assay. It is also possible though that our hypothesis is not correct and CHOP is not involved in UPR mediated apoptosis in these cells. Efforts are currently being made to optimize and sensitize the CHOP expression assay to gain positive results.

In conclusion, if our hypothesis were true, and CHOP is found to be involved in the apoptotic pathway in granulocytic precursors experiencing the UPR caused by expression of mutant NE, it is possible that new, more effective treatments for SCN could be developed with such knowledge of the molecular pathogenesis of SCN.