Biology subjects

Chalfant, C. E.

Publications and source records attributed to Chalfant, C. E..

6 recordsLinked to original sources

bioRxiv2026

ER Stress-Induced beta-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Development

Type 1 diabetes (T1D) is a consequence of {beta}-cell death. ER stress precedes T1D onset and prolonged ER stress in {beta}-cells can lead to {beta}-cell apoptosis. We reported that lipid signaling generated by the Ca2+-independent phospholipase A2{beta} (iPLA2{beta}), encoded by Pla2g6, participates in ER stress-mediated {beta}-cell apoptosis. {beta}-Cell membranes are enriched in arachidonic acid containing glycerophospholipids and the iPLA2{beta} catalyzes the hydrolysis of arachidonic acid in ER stressed {beta}-cells. Metabolism of arachidonic acid leads to the generation of various proinflammatory lipids, raising the possibility that they contribute to ER stress and {beta}-cell death leading to T1D. However, molecular mechanisms by which such {beta}-cell-iPLA2{beta}-derived lipid (iDL) signaling contributes to {beta}-cell apoptosis are not understood. It is well known that ER stress-mediated {beta}-cell apoptosis is associated with induction of transcription factors, NF{kappa}B and STAT1. We report here that both induce Pla2g6 and, unexpectedly, we find that iPLA2{beta}, which lacks DNA-binding motifs, associates with NFkB, Stat1, and Pla2g6 promoter regions. Consistently, p65-NF{kappa}B and pSTAT1 induction is reduced with select inhibition or knockdown of iPLA2{beta}. Surprisingly, iPLA2{beta} expression is also reduced by select inhibition of iPLA2{beta}, raising the possibility of feedback regulation by iDLs. In support, we find that select iDLs, recognized to be proinflammatory, enhance association of iPLA2{beta} with Pla2g6, Nfkb, and Stat1 promoter regions leading to induction of all three gene products and {beta}-cell apoptosis. Our findings reveal previously unrecognized transcriptional regulation by iDL signaling and, iPLA2{beta} itself, that leads to gene products that promote {beta}-cell apoptosis. Analogous findings in human islets validate this mechanism raising the possibility that targeting select lipid signaling can reduce ER stress in {beta}-cells and ameliorate T1D development.

cell biology↗

bioRxiv2025

The long non-coding RNA, CyKILRb, augments oncogenic phenotypes via induction of PIK3R2 and activation of the PI3K/AKT axis

Our laboratory recently identified a novel long noncoding RNA termed CyKILR that has two splice variants with distinct cellular localizations and opposing roles in tumorigenesis. The cytoplasmic variant, CyKILRb (exon 3 exclusion), promotes tumorigenesis, whereas the nuclear variant, CyKILRa (exon 3 inclusion), functions as a tumor suppressor. In this study, the molecular mechanism of the tumorigenic role of CyKILRb was characterized. Specifically, deep RNA sequencing analysis revealed that CyKILRb regulated the PI3K/AKT signaling pathway to block downstream tumor suppressors. In particular, downregulation of CyKILRb induced the loss of PIK3R2, an activator of PI3K, as well as RPS6KB2 and GNB2, two implicated tumor promotors, with a concomitant increase in the tumor suppressors, CDKN1A (p21) and CDKN1B (p27). In contrast, CyKILRb ectopic expression produced the opposite effect, and suppression of either PIK3R2, PI3K or AKT attenuated CyKILRb-induced cell proliferation and clonogenic survival. CyKILRb negatively regulated CyKILRa expression, which was blocked by inhibition of either PI3K or AKT. PIK3R2 ectopic expression overcame the cellular effects of CyKILRb downregulation, but not PI3K or AKT inhibition orienting the signaling pathway from CyKILRb[->]{uparrow}PIK3R2[->]PI3K[->]AKT[->]{downarrow}CyKILRa[->]enhanced oncogenicity. These findings highlight the critical role of CyKILRb in tumorigenesis and define a novel feed-forward regulatory mechanism linked to alternative RNA splicing.

cancer biology↗

bioRxiv2025

Acid ceramidase inhibition enhances BCL-2 targeting in venetoclax-resistant acute myeloid leukemia via a cytotoxic integrated stress response

Resistance to combination regimens containing the BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) is a growing clinical challenge for this extensively utilized agent. We previously established the anti-leukemic properties of ceramide, a tumor-suppressive sphingolipid, in AML and demonstrated that upregulated expression of acid ceramidase (AC), a ceramide-neutralizing enzyme, supported leukemic survival and resistance to BH3 mimetics. Here, we report the anti-leukemic efficacy and mechanisms of co-targeting AC and BCL-2 in venetoclax-resistant AML. Analysis of the BeatAML dataset revealed a positive relationship between increased AC gene expression and venetoclax resistance. Targeting AC enhanced single-agent venetoclax cytotoxicity and the venetoclax + cytarabine combination in AML cell lines with primary or acquired venetoclax resistance. SACLAC + venetoclax was equipotent to the combination of venetoclax + cytarabine at reducing cell viability when evaluated ex vivo across a cohort of 71 primary AML patient samples. Mechanistically, SACLAC + venetoclax increased ceramide to levels that trigger a cytotoxic integrated stress response (ISR), ISR-mediated NOXA protein upregulation, mitochondrial dysregulation, and caspase-dependent cell death. Collectively, these data demonstrate the efficacy of co-targeting AC and BCL-2 in AML and rationalize targeting AC as a therapeutic approach to overcome venetoclax resistance.

cancer biology↗

bioRxiv2024

RNA splicing variants of the novel long non-coding RNA, CyKILR, possess divergent biological functions in non-small cell lung cancer

The CDKN2A gene, responsible for encoding the tumor suppressors p16(INK4A) and p14(ARF), is frequently inactivated in non-small cell lung cancer (NSCLC). Herein, an uncharacterized long non-coding RNA (lncRNA) (ENSG00000267053) on chromosome 19p13.12 was found to be overexpressed in NSCLC cells with an active, wild-type CDKN2A gene. This lncRNA, named Cyclin-Dependent Kinase Inhibitor 2A-regulated lncRNA (CyKILR), also correlated with an active WT STK11 gene, which encodes the tumor suppressor, Liver kinase B1. CyKILR displayed two splice variants, CyKILRa (exon 3 included) and CyKILRb (exon 3 excluded), which are cooperatively regulated by CDKN2A and STK11 as knockdown of both tumor suppressor genes was required to induce a significant loss of exon 3 inclusion in mature CyKILR RNA. CyKILRa localized to the nucleus, and its downregulation using antisense RNA oligonucleotides enhanced cellular proliferation, migration, clonogenic survival, and tumor incidence. In contrast, CyKILRb localized to the cytoplasm, and its downregulation using siRNA reduced cell proliferation, migration, clonogenic survival, and tumor incidence. Transcriptomics analyses revealed enhancement of apoptotic pathways with concomitant suppression of key cell cycle pathways by CyKILRa demonstrating its tumor-suppressive role. CyKILRb inhibited tumor suppressor microRNAs indicating an oncogenic nature. These findings elucidate the intricate roles of lncRNAs in cell signaling and tumorigenesis.

molecular biology↗

bioRxiv2023

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell death-promoting signaling lipid that plays a central role in therapy-induced cell death. Acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug, LCL-805, across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 M was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 M was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

cancer biology↗

bioRxiv2020

The role of cytosolic polyadenylation element binding protein 2 alternative splicing in hypoxia

HIF1 (Hypoxia-inducible Factor 1) is a transcription factor that plays a crucial role in the hypoxia stress response. However, chronic hypoxia exposure can cause irreversible physiological changes that can lead to pulmonary hypertension (PH), and the need for therapeutics to ameliorate these conditions is great and unmet. Previous studies in our lab have demonstrated that CPEB2 (cytoplasmic polyadenylation element binding protein 2) is a translational repressor of one of the HIF1 subunits: HIF1. Our lab demonstrated that the alternatively spliced CPEB2A isoform of CPEB2 is a repressor of translation, while the CPEB2B isoform is a translational activator of HIF1 during hypoxia, suggesting a major regulatory role for CPEB2 AS in the pulmonary hypoxic response. Although it is well established that during hypoxia, HIF1 levels are dramatically upregulated due to a decrease in the degradation of this factor, we propose that during chronic hypoxia, the expression of HIF1 is maintained via a translational mechanism, likely alongside a decrease in proteolytic degradation. In this study we demonstrate that depletion of the CPEB2B splice isoform has an inhibitory effect on the translation of nascent HIF1 protein during chronic hypoxia, but not the acute phase. We further demonstrate this pathway is dependent on the initiation factor eIF3H. Finally, we show data that indicate CPEB2A and CPEB2B bind differentially to cytoplasmic polyadenylation element consensus sequences depending on the surrounding sequence context. These findings are important since they provide evidence for the potential of CPEB2 to act as a therapeutic target for treating chronic hypoxia-related pulmonary diseases.

cell biology↗