Biology subjects

Porter, S. N.

Publications and source records attributed to Porter, S. N..

5 recordsLinked to original sources

bioRxiv2026

Myogenic and neural transcriptional circuitry determines PAX3::FOXO1 driven initiation of rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) is largely characterized by the PAX3::FOXO1 (P3F) fusion oncoprotein, yet the mechanisms by which P3F initiates tumorigenesis and cell identity remain incompletely understood. Here, we describe a doxycycline inducible, degradable PAX3::FOXO1 iPSC derived ARMS model (ddP3F) to interrogate the temporal requirements of P3F for ARMS cell identity and proliferation. We show that, while P3F is essential for initiating ARMS transformation from precursor cells, continued expression of P3F is not required for maintaining proliferation, viability, or RMS associated transcriptional programs. Integration of CUT&RUN, bulk RNA seq, and 10X multiome profiling during endothelial-directed differentiation revealed that P3F rapidly pioneers a novel enhancer landscape within two days of expression, activating transcriptional circuits anchored by both specific myogenic and neural transcription factors (TFs), including MYOD1, PRDM12, and POU4F1 (BRN3A). Our single-cell analysis shows that these TFs are specifically co-expressed with P3F from the earliest stages of transformation. Knockout of MYOD1 or POU4F1 impairs ARMS identity acquisition without preventing transformation, whereas PRDM12 loss enhances P3F expression programs and myogenic differentiation. Together, these findings underscore a previously unappreciated interplay between myogenic and neural transcriptional programs during ARMS initiation. Furthermore, these data demonstrate that P3F establishes a stable oncogenic state that persists even after removal, thus providing a framework for evaluating P3F dependent and P3F independent therapeutic vulnerabilities.

cancer biology↗

bioRxiv2023

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for {beta}-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

genetics↗

bioRxiv2020

Interrogation of SLFN11 in pediatric sarcomas uncovers an unexpected biological role and a novel therapeutic approach to overcoming resistance to replicative stress

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress, and SLFN11 gene silencing has been implicated as a common mechanism of drug resistance in tumors in adults. We found SLFN11 to be widely expressed in our cohort of pediatric sarcomas. In sarcoma cell lines, protein expression strongly correlated with response to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN), with SLFN11 knockout resulting in significant loss of sensitivity in vitro and in vivo. However, SLFN11 expression was not associated with favorable outcomes in a retrospective analysis of our patient cohort; instead, the protein was retained and promoted tumor growth and evasion. Furthermore, we show that pediatric sarcomas develop resistance to TAL and IRN through impaired intrinsic apoptosis, and that resistance can be reversed by selective inhibition of BCL-XL. Statement of SignificanceThe role of SLFN11 in pediatric sarcomas has not been thoroughly explored. In contrast to its activity in adult tumors, SLFN11 did not predict favorable outcomes in pediatric patients, was not silenced, and promoted tumor growth. Resistance to replicative stress in SLFN11-expressing sarcomas was reversed by selective inhibition of BCL-XL.

cancer biology↗

bioRxiv2020

Za2 domain of ZBP1 is a molecular switch regulating Influenza-induced PANoptosis and perinatal lethality during development

Z-DNA-binding protein 1 (ZBP1) is an innate nucleic acid sensor which regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating RIPK3, caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate sensors because of its N-terminal Z1 and Z2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Z domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we have generated Zbp1{Delta}Z2/{Delta}Z2 mice that lack the Z2 domain of ZBP1 and demonstrate that this domain is critical for influenza A virus (IAV)-induced PANoptosis and perinatal lethality in RIPK1-RHIM mutated (Ripk1RHIM/RHIM) mice. Deletion of the Z2 domain in ZBP1 abolished IAV-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Z2 domain of ZBP1 was sufficient to rescue Ripk1RHIM/RHIM mice from the perinatal lethality which is caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Z2 domain of ZBP1 in physiological functions and establish a link between sensing of Z-RNAs via the Z2 domain and the promotion of influenza-induced PANoptosis and perinatal lethality.

immunology↗

bioRxiv2020

Enhanced stress tolerance through reduction of G3BP and suppression of stress granules

Stress granules (SG) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SG act as a protective mechanism to cope with acute stress, but persistent SG have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Importantly, knockout of the MAGE-B2 mouse ortholog confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermo-sensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress through fine-tuning phase separation and could aid in the development of male fertility therapies.

cell biology↗