Cohesin-mediated DNA loop extrusion enables gene regulation by distal enhancers through the establishment of chromosome structure and long-range enhancer-promoter interactions. The best characterized cohesin-related structures, such as topologically associating domains (TADs) anchored at convergent CTCF binding sites, represent static conformations. Consequently, loop extrusion dynamics remain poorly understood. To better characterize static and dynamically extruding chromatin loop structures, we use MNase-based 3D genome assays to simultaneously determine CTCF and cohesin localization as well as the 3D contacts they mediate. Here we present CTCF Analyzer (with) Multinomial Estimation (CAMEL), a tool that identifies CTCF footprints at near base-pair resolution in CTCF MNase HiChiP. We also use Region Capture Micro-C to identify a CTCF-adjacent footprint that is attributed to cohesin occupancy. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF loop) state is rare genome-wide with locus-specific variation from ~1–10%. We further investigate the impact of chromatin state on loop extrusion dynamics and find that active regulatory elements impede cohesin extrusion. These findings support a model of topological regulation whereby the transient, partially extruded state facilitates enhancer-promoter contacts that can regulate transcription.

Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical trials of EGFR inhibitors have yielded disappointing results. Using a candidate drug screen, we identified that inhibition of cyclin-dependent kinases 12 and 13 (CDK12/13) dramatically sensitizes diverse models of TNBC to EGFR blockade. This combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and translation-linked turnover of driver oncoproteins, including MYC. A genome-wide CRISPR/Cas9 screen identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, thereby rescuing MYC translational suppression and promoting MYC stability. The central roles of CCR4-NOT and 4E-BP1 in response to the combination therapy were further underscored by the observation of CNOT1 loss and rescue of 4E-BP1 phosphorylation in TNBC cells that naturally evolved therapy resistance. Thus, pharmacological inhibition of CDK12/13 reveals a long-proposed EGFR dependence in TNBC that functions through the cooperative regulation of translation-coupled oncoprotein stability.

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Background/Objectives: Randomized controlled trials (RCTs) are needed to evaluate the impact of food is medicine (FIM) programs, such as medically tailored groceries (MTGs) to treat hypertension among diverse populations. Partnerships between academic centers’ student-run organizations (SROs) and community-based organizations (CBOs) offer critical safety nets for historically underserved groups, positioning these organizations to effectively undertake FIM programs among populations disproportionately affected by hypertension. We conducted an unblinded pilot RCT whose objectives were to assess the feasibility and acceptability of an SRO-coordinated, CBO-partnered MTGs intervention targeting blood pressure (BP) and food insecurity (FI) in underserved groups. Methods: Adult Black/African American and Hispanic/Latinx patients in Durham, North Carolina, where essential hypertension and FI were randomized (parallel arm, computerized 1:1 ratio) to 12 weeks of home-delivered, hypertension-focused MTGs plus in-person nutrition education sessions with compensation (intervention) versus data collection sessions with compensation (control). We offered transportation, childcare, and home visits to facilitate session attendance. The primary outcomes were the eligibility, enrollment, and retention rates (feasibility), and the survey feedback from the participants and CBO partners (acceptability). The secondary outcomes included the changes in the mean BP and median FI score with associated 95% confidence intervals. Results: Medical record screening identified 1577 eligible participants. Of the 94 reached to confirm eligibility, 77 met the enrollment criteria, and 50 were randomized (82% post-screen eligibility, 65% enrollment). A conventional content analysis of 15 participant surveys and CBO partner feedback affirmed the acceptability, noting intervention components that enhanced the retention (e.g., home delivery, transportation support, home visits). Pre–post analyses of secondary outcomes for 13/25 intervention and 15/25 control participants completing ≥2 sessions ≥2 months apart were performed. The intervention was associated with an average change in systolic BP of −14.2 mmHg (−27.5, −4.5) versus −3.5 mmHg (−11.7, 5.9) in the control group. The FI scores improved by −2 (−2.2, −0.5) in the intervention group and −1 (−1.3, −0.2) in the control group. No adverse events were reported. Conclusions: SRO-CBO partnerships could be feasible and acceptable avenues for conducting FIM trials among underserved populations. This multi-component FIM approach enhanced the study equity by addressing the participants’ disease-related social needs and warrants expansion into a powered RCT.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Targeted therapies have revolutionized cancer care. Unfortunately, most patients develop refractory, multifocal resistance to these therapies within a matter of months. Here, we demonstrate that the evolution of resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer endows cells with hypersensitivity to a PAINS-like small molecule, MCB-613. Systematic proteomic, functional genomic, and biochemical studies revealed that MCB-613 binds KEAP1 in a covalent, cysteine-independent fashion, acting as a divalent molecular bridge that relies upon lysine residues in the KEAP1 dimerization domain to join monomers of KEAP1 together. Oligomerization of KEAP1 by MCB-613 sets into motion a fatal cascade of KEAP1 dysfunction, ROS accumulation, and ATF4/CHOP-dependent cell death. Together, these findings demonstrate that diverse models of EGFR inhibitor-resistant NSCLC share the common feature of elevated integrated stress response activity, and that a covalent molecular bridge which activates non-canonical KEAP1-ATF4 signaling can exploit this feature to select against resistance evolution.