Helen Piwnica-Worms

Chemotherapy remains the backbone of neoadjuvant treatment for triple-negative breast cancer (TNBC), and response to chemotherapy strongly correlates with patient prognosis. However, predicting chemoresistance remains a major clinical challenge. To address this obstacle, we performed serial fine needle aspirations followed by RNA-sequencing on patient-derived xenograft (PDX) models of TNBC that were either chemoresponsive or chemoresistant. We found that these two classes of PDX models exhibited distinct transcriptional trajectories that diverged within one week of chemotherapy initiation, preceding measurable changes in tumor volume. At early time points, chemoresponsive tumors showed activation of apoptotic and cytokine-related pathways, while these changes were absent in chemoresistant models. Notably, cytokine upregulation in chemoresponsive tumors coincided with infiltration of myeloid-derived immune cells into the tumor bed within seven days of chemotherapy, indicating active microenvironment remodeling, even in mice lacking a fully intact immune system. Analysis of paired biopsies from patients with TNBC collected before treatment and within four days of the first chemotherapy dose revealed a consistent pattern: chemoresponsive tumors upregulated inflammatory pathways, whereas chemoresistant tumors showed diminished activation of these pathways. These results underscore the potential clinical value of early on-treatment tumor sampling to detect chemoresistance and guide therapeutic intervention.

Fasting enhances small intestinal regeneration after radiation, but the contribution of the gut microbiome to this process remains uncharacterized. We identify Akkermansia muciniphila ( AKK ) as a key mediator of this response. AKK was enriched in fasted mice and its antibiotic depletion abrogated radioprotection, whereas reintroduction restored both organismal survival and intestinal integrity. Fasting elevated propionic acid, consistent with AKK ’s metabolic output. AKK -conditioned medium and propionate induced histone H3 acetylation in intestinal stem cell cultures while in vivo fasting induced AKK -dependent H3K27ac and H3K9ac, remodeling promoter–enhancer landscapes in crypt epithelial cells. Epigenetic profiling revealed a rewired core regulatory program enriched for pioneer transcription factors (Foxa, Gata, Klf), architectural organizers (Ctcf, Boris), and lineage-defining and metabolic regulators (Cdx2, Hnf4). This program supports expansion of a population of primed persister cells characterized by open chromatin accessibility at key stem and regenerative-associated loci including Clu , Olfm4 , Lgr5, Ascl2, Lrig1, Sox9, Rnf43, and Axin2. These findings define a fasting-induced microbiome–metabolite–chromatin axis that epigenetically primes highly plastic persister cells for rapid regeneration of the intestinal epithelium following radiation-induced injury.

Supplementary Table S1 shows relative IC50 of RSL3, ML210, JKE-1674, or ML162 in the indicated cells.

Supplementary Figure S1 shows that BRCA1 deficiency promotes GPX4i–induced ferroptosis. Supplementary Figure S2 shows that BRCA1 regulates GPX4 through the BRCT domain. Supplementary Figure S3 shows that BRCA1 deficiency suppresses erastin-induced ferroptosis via interference with VDAC3 transcription and mitochondrial lipid peroxidation.Supplementary Figure S4 shows that PARPi synergize with GPX4i in BRCA1-deficient cancers via ferroptosis. Supplementary Figure S5 shows that PARPi combined with GPX4i in BRCA1-mutant/deficient or homologous-recombination-restored cells or tumors. Supplementary Figure S6 shows that NCOA4-mediated ferritinophagy coupled with defective GPX4 induction contributes to the synergy of PARPi and GPX4i in BRCA1-deficient cancer cells. Supplementary Figure S7 shows that GPX4i overcome resistance to PARPi in BRCA1-mutant tumors.

Supplementary table S2 shows relevant gene mutation information for the cell lines and xenografts

Supplementary Table S3 shows sgRNA oligos sequences and primer sequences

Intrinsic and acquired resistance to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) remains a major barrier in treating homologous recombination (HR) repair-deficient tumors, including those with germline or somatic BRCA1/2 mutations. Although PARPi are FDA approved for adjuvant treatment of locally advanced or metastatic breast cancer in patients with germline BRCA1/2 mutations, emerging data support their use as monotherapy in the neoadjuvant setting. Promising safety profiles of newer-generation PARPi further support this potential. However, resistance mechanisms specific to the neoadjuvant setting are poorly understood. To address this gap, we leveraged resources from a phase II neoadjuvant clinical trial (NCT03499353), analyzing tumors from patients with germline BRCA1/2 mutant breast tumors before and after six months of talazoparib monotherapy. Whole-transcriptome analyses were performed on these samples. Additionally, we established orthotopic patient-derived xenograft models from a subset of the patient tumors and conducted whole-exome and whole-transcriptome analysis. This integrative approach revealed both known and previously unknown PARPi resistance mechanisms. In one case, overexpression of BRN2 , encoding a transcription factor that plays a critical role in neurogenesis, led to activation of ATR/RAD51 and STAT3 pathways, restoring HR repair. BRN2-driven resistance could be reversed with ATR and STAT3 inhibitors, resensitizing cells to talazoparib. In another, an HR repair proficient tumor subclone lacking Shieldin 2 expression expanded during treatment and accounted for intrinsic resistance. Our findings highlight the need to determine intrinsic and anticipate acquired resistance pathways in treatment-naïve tumors and support combining PARPi with targeted agents to improve outcomes in the neoadjuvant setting.

Systemic neoadjuvant chemotherapy, often combined with immunotherapy, is the standard of care for early-stage, non–breast cancer susceptibility gene (BRCA)-mutant triple negative breast cancer (TNBC). However, up to 70% of patients retain residual disease after treatment, which is linked to recurrence and mortality within 5 years. To define mechanisms of resistance, we performed single-cell RNA sequencing on orthotopic TNBC patient-derived xenografts during a cycle of treatment with doxorubicin and cyclophosphamide (AC). Clustering identified four tumor epithelial cell populations, with basal cells enriched in residual tumors. These basal cells up-regulated C15ORF48, a paralog of the mitochondrial cytochrome c oxidase associated subunit FA4 (NDUFA4), while exhibiting reciprocal down-regulation of NDUFA4. Functionally, C15ORF48 knockdown sensitized breast cancer cells to AC, increasing reactive oxygen species (ROS) and apoptosis. Thus, the up-regulation of C15ORF48 blunts ROS accumulation and induces resistance to chemotherapy in the basal cell subpopulations. Our findings identify C15ORF48 as a potential therapeutic target for overcoming AC resistance in TNBC.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes. In breast cancer, increased tumor stiffness is associated with elevated metastasis risks and poor survival. Here we report a unique role of the JAK family kinase TYK2 in suppressing breast cancer metastasis under low ECM stiffness. Genetic or pharmacological inhibition of TYK2 in mammary acini and patient-derived organoids leads to invasion at low stiffness by promoting Epithelial-Mesenchymal Transition, which is independent of cytokine-induced JAK/STAT signaling. TYK2 blockade promotes metastasis in breast tumor cell- and patient-derived xenografts. TYK2 localizes at the plasma membrane via IFNAR1 association under low ECM stiffness, whereas high rigidity causes TYK2 cytoplasmic mislocalization and inactivation. Consistently, normal breast epithelium displays membrane-localized TYK2, whereas invasive breast tumors exhibit cytoplasmic TYK2. These findings uncover a TYK2-dependent mechanism by which ECM rigidity suppresses breast cancer metastasis and underscore the need for breast cancer screening in patients receiving TYK2 inhibitors.

Fasting enhances small intestinal regeneration after radiation but the contribution of the gut microbiome to this process remains uncharacterized. We identify Akkermansia muciniphila ( AKK ) as a key mediator of this response. AKK was enriched in fasted mice and its antibiotic depletion abrogated radioprotection whereas reintroduction restored both organismal survival and intestinal integrity. Fasting elevated propionic acid, consistent with AKK ’s metabolic output. AKK -conditioned medium and propionate induced histone H3 acetylation in intestinal stem cell cultures while in vivo fasting induced AKK -dependent H3K27ac and H3K9ac, remodeling promoter-enhancer landscapes in crypt epithelial cells. Epigenetic profiling revealed a rewired core regulatory program enriched for pioneer transcription factors (Foxa, Gata, Klf), architectural organizers (Ctcf, Boris), and lineage-defining and metabolic regulators (Cdx2, Hnf4). This program supports expansion of a population of persister stem cells characterized by open chromatin accessibility at key stem and regenerative-associated loci including Clu , Olfm4 , Lgr5, Ascl2, Lrig1, Sox9, Rnf43, and Axin2. These findings define a fasting-induced microbiome-metabolite-chromatin axis that epigenetically primes highly plastic persister stem cells for rapid regeneration of the intestinal epithelium following radiation-induced injury. Significance Statement: Fasting changes the gut microbiome, but how these changes help the body recover from damage is not well understood. We found that fasting increases a helpful bacterium, Akkermansia muciniphila , which produces propionate, which drives epigenetic changes by modifying histones and regulating gene activity. These changes promote the expansion of persister stem cells that help the intestine recover after radiation. This study shows how fasting and gut bacteria work together to protect healthy tissue and suggests that diet or microbial treatments could help reduce side effects of cancer radiotherapy.