Biology subjects

Mulugeta, N.

Publications and source records attributed to Mulugeta, N..

6 recordsLinked to original sources

bioRxiv2025

Caspase Cleavage of Kaposi Sarcoma-Associated Herpesvirus Proteins: A role for K5 in Preventing Caspase-Mediated Cell Death during Lytic Replication

We previously reported that Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) acts as a pseudo-substrate for caspases-1 and 3, thereby interfering with their inflammatory and apoptotic activity, respectively. To determine if other KSHV proteins undergo caspase cleavage, we screened the KSHV proteome for potential caspase cleavage sites. Using SitePrediction (SP), 30 KSHV proteins with potential caspase-cleavage sites were identified. Among those with highest SP score was an early lytic protein, K5. Treatment of BJAB K5-FLAG cells with Fas, an apoptotic stimulus, led to caspase-processing of full length K5-FLAG and generation of a C-terminal peptide fragment. Using mass spectrometry, we determined that K5-FLAG undergoes caspase cleavage at D222. K5 was also cleaved by caspases in KSHV infected cells induced to lytic replication. Importantly, the expression of K5-FLAG significantly inhibited Fas-induced caspase-mediated cell death. To determine if K5 plays a protective role in KSHV infected cells, iSLKK cells infected with wild type or K5 knockout BAC16 virus were induce to lytic replication to activate caspases. Although lytic induction showed little effect on the viability of WT infected cells, the viability of K5-knockout cells decreased by 25%. Thus, K5 may protect KSHV-infected cells from caspase-mediated cell death during lytic replication. Interestingly, cleavage of K5 by caspases did not affect its ability to downregulate MHC-1 surface expression. Overall, these data suggest that K5 not only downregulates immunologic surface marker expression to avoid immune recognition but may also play an additional role in mitigating caspase-mediated cell death during KSHV lytic replication.

biochemistry↗

bioRxiv2024

Mutation and cell state compatibility is required and targetable in Ph+ acute lymphoblastic leukemia minimal residual disease

Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples. HIGHLIGHTSO_LIRelapse after remission on TKI can harbor mutations in ABL1, RAS, or neither C_LIO_LIMutations and development-like cell state dictate fitness in residual disease C_LIO_LICo-targeting cell state and ABL1 markedly reduces MRD C_LIO_LIBiophysical measurements provide an integrative, rapid measurement of cell state C_LI

cancer biology↗

bioRxiv2024

Exposure to HIV alters the composition of maternal microchimeric T cells in infants

Infants exposed to HIV but uninfected (iHEU) display altered cellular immunity and are at increased risk of infection through poorly understood mechanisms. We previously reported that iHEU have lower levels of maternal microchimerism (MMc), maternal cells transferred to the offspring in utero/during breastfeeding. We evaluated MMc levels in T cell subsets in iHEU and HIV unexposed infants (iHU) to determine whether a selective deficiency in MMc may contribute to altered cellular immunity. Across all infants, MMc levels were highest in CD8+ T cells; however, the level of MMc in the CD8 T cell subset was significantly lower in iHEU compared to iHU.

immunology↗

bioRxiv2021

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation in severe disease

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth-stunting, and diminished oral vaccine responses. While EE has been shown to be the by-product of recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE severity by performing high-throughput single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE from Lusaka, Zambia (8 HIV-negative, 3 HIV-positive), 6 adults without EE in Boston, USA, and 2 adults from Durban, South Africa, which we complemented with published data from 3 additional South African adults from the same clinical site. By using these data to reanalyze previously-defined bulk-transcriptomic signatures of reduced villus height and decreased plasma LPS levels in EE, we found that these signatures may be driven by an increased abundance of surface mucosal cells - a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we identified several cell subsets whose fractional abundances associate with histologically determined EE severity, small intestinal region, and HIV infection. Furthermore, by comparing distal duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells but with increased pro-inflammatory cytokine expression in the EE cohort. Altogether, our work illuminates epithelial and immune correlates of EE and provides new molecular targets for intervention. One Sentence SummaryUsing single-cell RNA-sequencing, we characterize the pathophysiology of environmental enteropathy (EE) - a highly prevalent condition of the small intestine that is thought to be a primary cause of global growth-stunting cases and a key contributing factor to childhood malnutrition and diminished oral vaccine responses - to derive insights into the epithelial and immune correlates of disease severity, suggesting new therapeutic targets for future investigation.

genomics↗

bioRxiv2020

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice

Existing pre-clinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates using an autochthonous small cell lung cancer model, we extrapolated half-life times in the circulation of 50-100 seconds and intravasation rates between 4,000 and 27,000 CTCs/hour - an average daily shedding rate equivalent to [~]0.07% of the total number of primary tumor cells in the lung. Additionally, transfer of 1-2% of daily-shed CTCs from late-stage tumor-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

cancer biology↗

bioRxiv2020

Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer

Bulk transcriptomic studies have defined classical and basal-like gene expression subtypes in pancreatic ductal adenocarcinoma (PDAC) that correlate with survival and response to chemotherapy; however, the underlying mechanisms that govern these subtypes and their heterogeneity remain elusive. Here, we performed single-cell RNA-sequencing of 23 metastatic PDAC needle biopsies and matched organoid models to understand how tumor cell-intrinsic features and extrinsic factors in the tumor microenvironment (TME) shape PDAC cancer cell phenotypes. We identify a novel cancer cell state that co-expresses basal-like and classical signatures, demonstrates upregulation of developmental and KRAS-driven gene expression programs, and represents a transitional intermediate between the basal-like and classical poles. Further, we observe structure to the metastatic TME supporting a model whereby reciprocal intercellular signaling shapes the local microenvironment and influences cancer cell transcriptional subtypes. In organoid culture, we find that transcriptional phenotypes are plastic and strongly skew toward the classical expression state, irrespective of genotype. Moreover, we show that patient-relevant transcriptional heterogeneity can be rescued by supplementing organoid media with factors found in the TME in a subtype-specific manner. Collectively, our study demonstrates that distinct microenvironmental signals are critical regulators of clinically relevant PDAC transcriptional states and their plasticity, identifies the necessity for considering the TME in cancer modeling efforts, and provides a generalizable approach for delineating the cell-intrinsic versus -extrinsic factors that govern tumor cell phenotypes.

cancer biology↗