Abstract The tumor microenvironment (TME) comprises diverse cell types, including mesenchymal, endothelial, adipocyte, stromal, and immune cells. Among them, cancer-associated fibroblasts (CAFs) represent one of the most abundant and functionally active components. CAFs play a crucial role in tumor progression through bidirectional communication with cancer cells. They originate from various sources, such as normal fibroblasts, endothelial cells, and mesenchymal cells, and once activated, promote tumor cell invasion and metastasis. Recent studies have revealed that CAFs consist of multiple subpopulations with distinct phenotypic and functional properties. This heterogeneity of CAFs has provided new insights into tumor biology and has become a key focus in the development of novel targeted therapeutic strategies across different cancer types. CRISPR/Cas9 is a powerful genome-editing tool widely used for knock-out (KO) gene studies to investigate gene function. To overcome the limitations of conventional phenotyping and bulk analysis, a barcoding system known as Perturb-map was developed by the Brown laboratory that allows KO of genes in tumor cells and facilitates identification of effects of the KO on the TME. Perturb-map utilizes triplet combinations of linear epitope protein barcodes (Pro-codes) that enable the identification of cells expressing distinct CRISPR guide RNAs (gRNAs). In this study, we applied the Perturb-map approach to perform parallel CRISPR KO of 34 genes closely associated with CAF function in the TME in a syngeneic mouse breast cancer model. This approach allowed us to simultaneously assess the functional roles of multiple TME-related genes in tumor cells, providing a comprehensive understanding of their contributions to tumor progression. Pro-code-expressing tumors were analyzed using cyclic immunofluorescence (CycIF), a highly multiplexed proteomics imaging platform that enables spatial and single-cell level analysis. We developed, validated, and applied mouse antibody panels targeting approximately 100 proteins, allowing comprehensive profiling of tumor heterogeneity, cellular states, fibroblast, and immune cell activities. Through this approach, we identified potential therapeutic targets in tumor cells that confer growth advantages and contribute to remodeling of the TME. Furthermore, we identified effects of the KOs on CAF subtypes with distinct functional states. CD274 and IL11Rα1 KO tumors exhibited accelerated tumor growth accompanied by an increased abundance of myofibroblastic CAFs (myCAFs). In contrast, Snai2 KO tumors showed a marked enrichment of inflammatory CAFs (iCAFs). Our findings demonstrate the power of integrating functional genomics with high-dimensional proteomics to characterize TME dynamics at single-cell resolution. Citation Format: Boyoung Jeong, Xuejiao Zhao, David Kilburn, Kang Jin Jeong, Soon Young Park, Hongli Ma, Gordon B. Mills. Single-cell spatial CRISPR screen for tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6200.

Abstract BACKGROUND: The poly(ADP-ribose) polymerase-1 (PARP1) enzyme is critical to DNA Damage Response. Multiple first generation non-selective, trapping PARP1/2 inhibitors have been successful as single agent cancer treatments in homologous recombination (HR)-deficient malignancies; on the other hand, first-generation PARPi such as olaparib (a strong PARP1 trapper), or veliparib (a PARP1 trapper with medium potency), have not been successful in combination with chemotherapy due to a poor therapeutic window. Recently, second generation trapper PARP1-selective inhibitors (e.g. saruparib, M9466, SNV1521 and palacaparib) have entered clinical development, based on preclinical evidence that PARP2 inhibition is associated with anemia and dispensable for anti-tumor efficacy. However, emerging clinical data suggest that these drugs display hematological effects similar to the first generation PARPi. Indeed, all these inhibitors trap PARP1 on DNA lesions, causing hematological adverse effects that are overlapping with those caused by chemotherapy agents such as temozolomide (TMZ) and topoisomerase I inhibitors (TOP1i), thus limiting their combination potential. METHODS: We used biochemical and cellular assays to evaluate itareparib potency and selectivity. Additionally, we performed cellular proliferation assays and mechanistic assays such as DNA damage markers assessment by high-content imaging and DNA combing assays, to evaluate itareparib activity in combination with DNA damaging agents (DDA). Finally, we performed in vivo experiments in tumor bearing mice and tumor free rats to assess the combination effects of itareparib plus TMZ on tumor growth inhibition and on the bone marrow. RESULTS: Itareparib is a non-trapper PARPi and showed synergistic activity in combination with TMZ and TOP1i or Antibody Drug Conjugates (ADCs) with TOP1i payloads on DNA damage markers and in proliferation assays in vitro. The combination efficacy with temozolomide was confirmed in vivo in a small cell lung cancer xenografts model. Additionally, the comparative data with trapper-PARPi suggests that the combination efficacy with TMZ and TOP1i is not dependent on the trapping potency. Importantly, itareparib combination with DDA had lower impact on bone marrow precursors both in vitro and in vivo compared to PARP1-trapping inhibitors. Consistently with literature data, in vitro studies identify SLFN11 expression as a sensitivity biomarker to the combination of itareparib with chemotherapy. CONCLUSIONS: Itareparib features are designed to expand the application of PARP1i through combination with chemotherapy, ADCs and radionuclides addressing the unmet need of patients with both HR-deficient and HR-proficient tumors. Itareparib is currently in Phase I/II clinical trials in combination with DNA-damaging agents in brain (high grade gliomas), lung and ovarian cancer. Citation Format: Alessandro Galbiati, Gianluca Papeo, Nilla Avanzi, Fabio Gasparri, Claudia Perrera, Gemma Texido, Lisa Mahnke, Shiho Nakano, Kang Jin Jeong, Gordon B. Mills, Junko Murai, Alessia Montagnoli. Itareparib: A potent, selective and non-trapper PARP1 inhibitor for combination therapy with DNA damaging agents in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2933.

Gemcitabine, a ribonucleotide reductase (RNR) inhibitor, is active in pancreatic ductal carcinoma (PDAC) patients, but unfortunately has a limited impact on long term outcomes. Gemcitabine induces nucleotide deficiency, DNA damage including single stranded DNA (ssDNA) and replication stress (RS). DNA damage can activate cyclic GMP-AMP synthase (cGAS), leading to genome instability, micronucleus generation, and immune activation. In model systems, gemcitabine resistance can be overcome by combination treatment with the ataxia telangiectasia and Rad3-related inhibitor (ATRi; AZD6738) that blocks S and G2 checkpoints, although underlying mechanisms remain to be fully elucidated. We show that cells with low basal RS are resistant to gemcitabine, which could be overcome by combination treatment with AZD6738 through elevation of RS, phospho-RPA32 exhaustion, and mitotic catastrophe in PDAC cell models. Gemcitabine induces nuclear cGAS accumulation independent of STING-mediated immune activation. The binding of nuclear cGAS to γH2AX at double strand DNA breaks (DSBs) plays a pivotal role in RS activation and mitotic catastrophe in gemcitabine and AZD6738 treated cells.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease soon to become the second leading cause of cancer deaths in the US. Beside surgery, current therapies have narrow clinical benefits with systemic toxicities. FOLFIRINOX is the current standard of care, one component of which is 5- Fluorouracil (5-FU), which causes serious gastrointestinal and hematopoietic toxicities and is vulnerable to resistance mechanisms. Recently, we have developed polymeric fluoropyrimidines (F10, CF10) which unlike 5-FU, are, in principle, completely converted to the thymidylate synthase inhibitory metabolite FdUMP, without generating appreciable levels of ribonucleotides that cause systemic toxicities while displaying much stronger anti-cancer activity. Here, we confirm the potency of CF10 and investigate enhancement of its efficacy through combination with inhibitors in vitro targeting replication stress, a hallmark of PDAC cells. CF10 is 308-times more potent as a single agent than 5-FU and was effective in the nM range in primary patient derived models. Further, we find that activity of CF10, but not 5-FU, is enhanced through combination with inhibitors of ATR and Wee1 that regulate the S and G2 DNA damage checkpoints and can be reversed by addition of dNTPs indicative of CF10 acting, at least in part, through inducing replication stress. Our results indicate CF10 has the potential to supersede the established benefit of 5-FU in PDAC treatment and indicate novel combination approaches that should be validated in vivo and may be beneficial in established regimens that include 5-FU.

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases, including BRCA1/2 mutant breast cancer, responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing, we identify a TAM_C3 cluster, expressing genes implicated in anti-inflammatory activity, that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity, respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus, targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies. PARP inhibitors (PARPi) have been approved for the treatment of metastatic triple-negative breast cancer (BC), however resistance and recurrence are often observed. Here, in preclinical models of BRCA1/2 wild type and homologous recombination competent BC, the authors show that C5aR1-positive tumor associated macrophages are associated with PARPi-resistance, suggesting targeting C5aR1 as a therapeutic option.

GAB2 mediates the effects of siYAP/TAZ on the PI3K/AKT pathway

Knockdown of POSTN expression decreases GSC11 tumor volume and invasion.

<p>The treatment effect of YAP/TAZ inhibitor on an EC animal model</p>

Histogram demonstrating distribution of (A) RAS, (B) KRAS, and (C) NRAS mutations across clinical cohorts with survival data based on activity level.

GAB2 mRNA is markedly altered by siYAP/TAZ

<p>siYAP/TAZ inhibits PI3K/AKT pathway activation</p>

Frequency and prevalence of KRAS mutations across 8 pooled cohorts

The effects of YAP/TAZ on biological behavior of EC cells

<p>siYAP/TAZ inhibits PI3K/AKT pathway activation</p>

<p>GAB2 mediates the effects of siYAP/TAZ on the PI3K/AKT pathway</p>

Western blot of RAS expression and accompanying RAS-GTP pulldown assay for isogenic SW48 cell lines generated for xenograft experiments.

Frequency and prevalence of NRAS mutations across 8 pooled cohorts

Summary of functional validation across four platforms for variants characterized by all assays.

Ferroptosis, a form of programed cell death, can be promoted by inhibitors of the xCT transporter (erastin) or GPX4 (RSL3). We found that GPX4, but not the xCT transporter, is selectively elevated in luminal breast cancer. Consistent with this observation, the majority of luminal breast cancer cell lines are exquisitely sensitive to RSL3 with limited sensitivity to erastin. In RSL3-resistant, but not sensitive, luminal breast cancer cell lines, RSL3 induces HER2 pathway activation. Irreversible HER2 inhibitors including neratinib reversed RSL3 resistance in constitutively RSL3-resistant cell lines. Combination treatment with RSL3 and neratinib increases ferroptosis through mitochondrial iron-dependent reactive oxygen species production and lipid peroxidation. RSL3 also activated replication stress and concomitant S phase and G2/M blockade leading to sensitivity to targeting the DNA damage checkpoint. Together, our data support the exploration of RSL3 combined with irreversible HER2 inhibitors in clinical trials.