Biology subjects

Stern, J. L.

Publications and source records attributed to Stern, J. L..

4 recordsLinked to original sources

bioRxiv2024

SNAI2 cooperates with MEK1/2 and HDACs to suppress BIM- and BMF-dependent apoptosis in TERT promoter mutant cancers

Cancers with TERT promoter mutations (TPM) display elevated RAS pathway signaling and mesenchymal traits, and associate with lower patient survival rates. We examined whether RAS pathway signaling in TPM cancers cooperates with mesenchymal features to drive resistance to apoptosis. We observed that RAS pathway signaling in TPM cancers inhibited apoptosis by downregulating the pro-apoptotic protein BIM. By using inhibitors of MEK1/2 kinases, we rescued the ability of TPM cancer cells to undergo apoptosis, which may have implications for targeted therapies. To further capitalize on this rescue, we explored combination treatments to drive apoptotic cell death. Treatment with the pan-BCL2 inhibitor, navitoclax (NX), in combination with MEK inhibition, significantly increased apoptosis, indicating that these cells are capable of undergoing intrinsic apoptosis, with BIM likely playing a critical role. Further, we found that transcriptional reprogramming of the mesenchymal state of TPM cancers using histone deacetylase inhibitors (HDACi) resulted in a synergistic increase in apoptosis, contingent upon BIM de-repression. Notably, the cause of this apoptosis appeared to be independent of DNA damage. The suppression of the mesenchymal transcription factor SNAI2, which has known roles in recruiting HDACs to silence gene expression, amplified apoptosis. Mechanistically, knockdown of SNAI2 impaired the cellular DNA repair leading to elevated basal levels of phosphorylated H2AX. Our findings show that TPM cancers exhibit specific small molecule vulnerabilities, driven by the convergence of RAS-MEK signaling and impaired HDAC regulation dependent on pro-apoptotic BH3-only proteins. Based on our findings, we propose that stratifying cancers based on TPM may identify a subset of tumors that are responsive to innovative combinations of inhibitors targeting these axes.

cancer biology↗

bioRxiv2024

Apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2/APE2) is required for efficient expression of TERT in human embryonic stem cells.

Human stem cells rely on enhanced DNA repair mechanisms to safeguard their ability to replenish somatic tissues. Telomerase counteracts telomere shortening and is a component of the stem cell DNA repair system that is regulated by ATM and ATR kinases. Here, we report that the DNA repair enzyme APEX2, but not its close paralog APEX1, is required for efficient telomerase reverse transcriptase (TERT) gene expression in human embryonic stem cells (hESC) and a melanoma cell line. We also observed that APEX2 knockdown significantly diminished telomerase enzyme activity. While APEX1 is known to regulate certain transcription factors, APEX2 has not been reported to influence gene expression. To gain insight into how APEX2 influences gene expression, we conducted RNA-seq following APEX2 knockdown in hESC. These results indicated that a number of genes, in addition to TERT, relied on APEX2 for efficient expression. Genes affected by APEX2 knockdown were significantly enriched for specific repetitive DNA families. These include mammalian-wide interspersed repeats (MIRs) and Alu elements. Chromatin immunoprecipitation experiments demonstrated the highest APEX2 binding near MIR sequences in TERT intron 2. Surprisingly, binding was low in the TERT proximal promoter, a region known to control TERT transcription. MIR and other repetitive DNA regions are common sites of DNA damage, suggesting that APEX2 recruitment and repair of TERT MIR sequences may play a role in influencing TERT expression. This new role for APEX2 in promoting efficient gene expression deepens our understanding of an emerging cancer therapeutic target. Further, as the TERT gene plays critical roles in stem cell maintenance, organismal development and aging, as well as in short telomere disorders and cancer, our observations provide insight into new strategies to modulate the expression of this important enzyme.

cell biology↗

bioRxiv2024

MEK1/2 kinases cooperate with c-Myc:MAX to prevent polycomb repression of TERT in human pluripotent stem cells

Telomerase counteracts telomere shortening, enabling human embryonic stem cells (hESC) to undergo long-term proliferation. MAPK signaling plays a major role in regulating the self-renewal of hESC, and previous studies in induced pluripotent stem cells (iPSC) suggested that expression of TERT, the gene encoding the catalytic subunit of telomerase, relies on MAPK signaling. We examined whether MEK-ERK signaling regulated TERT transcription in a model of normal hESC. Kinase inhibitors of MEK1 and MEK2 (MEKi) or ERK1 and ERK2 (ERKi) significantly repressed TERT mRNA levels. Using chromatin immunoprecipitation (ChIP) we observed that MEKi induced the accumulation of the repressive histone mark histone 3 lysine 27 trimethylation (H3K27me3) at the TERT proximal promoter. This increase corresponded with a loss of histone 3 lysine 27 acetylation (H3K27ac) which is associated with transcriptionally active loci. Inhibition of the polycomb repressive complex 2 (PRC2), which deposits H3K27me3, partially rescued the loss of TERT expression, indicating that MEK1/2 activity can limit PRC2 activity at TERT. Inhibition of MEK/ERK kinases also repressed expression of c-Myc, a transcription factor reported to regulate TERT in other immortalized cells. Consistent with a key role for c-Myc in regulating TERT, low doses of a c-Myc:MAX dimerization inhibitor induced a striking and rapid gain of H3K27me3 at TERT and repressed TERT transcription in hESC. Inhibiting c-Myc:MAX dimerization also resulted in lower MAX recruitment to TERT, suggesting that this complex acts in cis at TERT. Our study using a model of normal human pluripotent stem cells identifies new regulators and mechanisms controlling transcription of an important, developmentally regulated gene involved in telomere protection.

cell biology↗

bioRxiv2021

Epigenetic remodeling during monolayer cell expansion reduces therapeutic potential

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells. SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

bioengineering↗