PURPOSE:Translational Breast Cancer Research Consortium 048 was a proof-of-principle trial demonstrating responses to the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in patients (pts) with metastatic breast cancer (MBC) with germline (g) PALB2 or somatic (s) BRCA mutations (sBRCAm). Here we report results from the expansion cohorts in a larger sample of pts with gPALB2m or sBRCAm. METHODS:Eligible pts had MBC of any subtype with measurable disease and a gPALB2m or sBRCAm. Pts received olaparib 300 mg twice a day until progression. The primary end point was overall response rate. Secondary end points include clinical benefit rate (CBR) at 18 weeks, progression-free survival (PFS), duration of response (DOR), and whether among sBRCAm carriers the mutant allele frequency (MAF) is significantly higher in responders than in nonresponders. RESULTS:Fifty-four pts with gPALB2m (N = 24) or sBRCAm (N = 30) were enrolled. Forty-two (78%) had estrogen receptor-positive human epidermal growth factor receptor 2-negative (HER2-) MBC, seven (13%) had triple-negative breast cancer, and five (9%) had HER2+ disease. Among pts with a gPALB2m, the overall response rate (ORR) was 75% (80% CI, 60.2 to 86.3), CBR was 83.3% (90% CI, 65.8 to 94.1), the median PFS was 9.4 months (90% CI, 8.3 to 13.1), and the median DOR was 7.0 months (90% CI, 5.6 to 10.4). Among pts with sBRCAm (15 sBRCA1 and 15 sBRCA2), the ORR was 36.7% (80% CI, 24.7 to 50), CBR was 53.3% (90% CI, 37 to 69.1), the median PFS was 5.5 months (90% CI, 2.8 to 8.3), and the median DOR was 11.2 months (90% CI, 4.4 to not reached). One additional pt had an unconfirmed partial response. Although clinically meaningful, the ORR in pts with sBRCAm did not achieve the prespecified target. Among sBRCAm carriers, the mean MAF did not differ significantly between responders (46%) and nonresponders (39%; P = .7). CONCLUSION:Olaparib is active in pts with MBC with gPALB2m and sBRCAm, significantly expanding the population of pts with breast cancer likely to benefit from PARP inhibitors beyond gBRCA1/2m carriers.

Background:We investigated the impact of gender-affirming testosterone therapy (TT) on breast cancer (BC) risk and tumor progression. Materials and methods:We leveraged a large human breast tissue dataset (n=417) to assess TT and terminal duct lobular unit (TDLU) involution, complemented with tissue markers (ER, PR, AR, and Ki67; n =24) and transcriptome profiling ( n =8). Preclinical models assessed the effect of TT on BC incidence ( MMTV-Cre Pik3ca f/wt n =149 and K14-Cre Brca f/f Tp53 f/f n =153), murine mammary gland architecture ( n =60), and tumor transcriptome ( n =10). Lastly, we discuss trans masculine invasive BC cases and summarize tumor characteristics in this population ( n =24). Results:TT promotes TDLU involution by reducing epithelial proliferation via altered estrogen signaling and increases ER+, PR+, and Ki67+ extralobular stromal cells. In mice, TT similarly reduced mammary gland ductal branching and terminal end buds. TT decreased Pik3ca -related ER+ BC incidence by 81% compared to female controls (adj RR 0.19, 95% CI 0.08-0.45), but did not affect Brca1 -related triple negative BC incidence. TT did not influence tumor progression in either model but shaped the Pik3ca -related ER+ tumor microenvironment toward a pro-tumor phenotype. Most trans masculine BC cases were ER+ (83.3%), small and node-negative, but were also moderately to poorly differentiated (70.8%). Conclusion:TT reduces ER+ BC risk but does not eliminate risk, and has a negligible impact on BRCA1 -related triple-negative BC risk. TT does not affect tumor growth once tumors are established but modulates the tumor microenvironment. Our work supports the need for breast cancer screening in TT users. Highlights: TT reduces but does not completely ablate the breast epithelium. TT decreases PIK3CA -related ER+ breast cancer incidence by 81% compared to female control mice (adj RR 0.19, 95% CI 0.08-0.45), but does not affect BRCA1 -related triple negative breast cancer incidence. TT does not affect tumor progression once the tumor is established.Trans masculine breast tumors are mostly ER+ (83.3%), small and node-negative, but are also moderately to poorly differentiated (70.8%).Tailored risk assessment and ongoing surveillance strategies are key for the care of transmasculine individuals who use TT.

Homozygous MTAP deletion occurs in ~15% of cancers, making them vulnerable to decreases in the concentration of S-adenosylmethionine (SAM). AG-270/S095033 is an oral, potent, reversible inhibitor of methionine adenosyltransferase 2 A (MAT2A), the enzyme primarily responsible for the synthesis of SAM. We report results from the first-in-human, phase 1 trial of AG-270/S095033 as monotherapy in patients with advanced malignancies (ClinicalTrials.gov Identifier: NCT03435250). Eligible patients had tumors with homozygous deletion of CDKN2A/MTAP and/or loss of MTAP protein by immunohistochemistry. Patients received AG-270/S095033 once daily (QD) or twice daily (BID) in 28-day cycles. The primary objective was to assess the maximum tolerated dose (MTD) of AG-270/S095033. Secondary objectives included safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Forty patients were treated with AG-270/S095033. Plasma concentrations of AG-270/S095033 increased with dose. Maximal reductions in plasma SAM concentrations ranged from 54% to 70%. Analysis of paired tumor biopsies showed decreases in levels of symmetrically di-methylated arginine (SDMA) residues. Reversible increases in liver function tests, thrombocytopenia, anemia and fatigue were common treatment-related toxicities. Two partial responses were observed; five additional patients achieved radiographically confirmed stable disease for ≥16 weeks. AG-270/S095033 has a manageable safety profile. Our data provide preliminary evidence of clinical activity and proof-of-mechanism for MAT2A inhibition. AG-270 inhibited the activity of MAT2A and reduced plasma concentrations of SAM by up to 70% in patients with advanced solid tumors. Partial responses were observed in two patients and disease stabilization for ≥4 months was seen in five patients.

Combination chemotherapy remains essential for clinical management of triple-negative breast cancer (TNBC), making it impossible to assess responses to multiple agents in a single patient. Herein, we conduct multi-omic analyses of TNBC patient-derived xenografts (PDXs) treated with single agent carboplatin and docetaxel, or the combination, to identify candidate mechanisms of resistance, as well as predictive biomarkers to individual treatments, and to develop therapeutic strategies to overcome resistance. Genomic, transcriptomic, and proteomic profiles of baseline tumors from 50 TNBC PDXs were associated with responses to human equivalent doses of either single agent carboplatin, docetaxel, and the combination. Integration of external TNBC PDX and clinical datasets was performed using ComBat. Protein Marker Selection (ProMS) tool was used to integrate both RNA and protein data to select 5-10 RNA feature combinations for optimized prediction of chemotherapy response in a logistic regression model. Combination responses were generally no better than the best single agent, with enhanced response in only ∼13% of PDX, and apparent antagonism in a comparable percentage. Single ome comparisons showed largely non-overlapping results between genes associated with single agent and combination treatment that validated in independent patient cohorts. Multi-omic analyses of PDXs identified agent-specific treatment-associated biomarkers and predictive biomarker combinations. Notably, integrating proteomic with mRNA data improved machine learning models that achieved AUROC performance of 0.85 to predict pathologic complete response to combination chemotherapy. These models await further evaluation in datasets that may become available, including data from the BEAUTY (NCT02022202), TBCRC 030 (NCT01982448), and RESPONSE (NCT05020860) clinical trials, or in future experimental settings using PDX/PDX organoids. In PDXs responsive to any treatment, several basal cytokeratins were among the top upregulated proteins compared to PDXs resistant to all treatments. KRT5 was validated by IHC in PDX tumors, achieving an AUROC of 0.83 for predicting responsiveness, indicating its potential as a future chemoresponse marker in TNBC. PDXs refractory to all treatments showed dysregulated mitochondrial function. Treatment with romidepsin, an HDAC inhibitor that targets this process indirectly, increased DNA damage, and enhanced carboplatin response, in a chemoresistant PDX model with high abundance of HDAC proteins. Multi-omic characterization identifies molecular mechanisms and predictive biomarkers for stratifying TNBC tumors for single or combination chemotherapy treatments, suggests targeted therapies to augment chemotherapy response, and provides a valuable resource for researchers and clinicians. Jonathan T. Lei, Lacey E. Dobrolecki, Chen Huang, Ramakrishnan R. Srinivasan, Suhas V. Vasaikar, Alaina N. Lewis, Christina Sallas, Na Zhao, Jin Cao, John D. Landua, Chang I. Moon, Yuxing Liao, Susan G. Hilsenbeck, C K. Osborne, Mothaffar F. Rimawi, Matthew J. Ellis, Varduhi Petrosyan, Bo Wen, Kai Li, Alexander B. Saltzman, Antrix Jain, Anna Malovannaya, Gerburg M. Wulf, Elisabetta Marangoni, Shunqiang Li, Daniel C. Kraushaar, Tao Wang, Senthil Damodaran, Xiaofeng Zheng, Funda Meric-Bernstam, Gloria V. Echeverria, Meenakshi Anurag, Xi Chen, Bryan E. Welm, Alana L. Welm, Bing Zhang, Michael T. Lewis. Patient-derived xenografts allow deconvolution and prediction of chemotherapy responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 739.

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in three clusters, including an IMiD degron in zinc finger cluster one (ZFC1). Surprisingly, IMiDs do not inhibit the growth of SALL4-expressing cancer cells. To overcome this limit, we focused on a non-IMiD domain, SALL4 zinc finger cluster four (ZFC4). By combining ZFC4-DNA crystal structure and an in silico docking algorithm, in conjunction with cell viability assays, we screened several chemical libraries against a potentially druggable binding pocket, leading to the discovery of SH6, a compound that selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 treatment led to a significant 87% tumor growth inhibition of SALL4+ patient-derived xenografts and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors such as SALL4 in cancer.

3009 Background: B7-H4 is a transmembrane protein over-expressed in breast (BC), ovarian (OC), endometrial (EC), and adenoid cystic carcinoma type 1 (ACC-1) cancers, with limited expression in healthy tissues. Emi-Le (XMT-1660) is a B7-H4-directed Dolasynthen ADC designed with a proprietary auristatin F-HPA microtubule inhibitor payload with controlled bystander effect. Methods: The Phase 1 trial is investigating Emi-Le monotherapy in adult patients (pts) with advanced/metastatic TNBC, HR+/HER2- BC, OC, EC and ACC-1. In dose escalation, eligible pts received Emi-Le at doses of 7.2-115 mg/m2 per cycle, with all collected data informing the recommended doses for the expansion (EXP) portion of the trial. Tumors were evaluated retrospectively for B7-H4 expression by IHC, with the preliminary high cutoff set at TPS≥70. Results: As of December 13, 2024, 130 pts were dosed. Across all tumor types, median age of pts was 55; median 4.5 prior lines of therapy (range 0-15). B7-H4 status was evaluated for 103 pts, with 44% determined to be B7-H4 TPS high. Overall, Emi-Le was generally well tolerated. The most common TRAEs were transient AST increase (38%, G3 14%), proteinuria (31%, G3 9%), nausea (29%, G3 1%) and fatigue (28%, G3 0%). The only G3 TRAEs in ≥5% of pts were AST increase and proteinuria. No G4 or 5 TRAEs were reported. No observed dose-limiting treatment-related neutropenia, neuropathy, ocular toxicity, interstitial lung disease or thrombocytopenia. TRAEs leading to discontinuation were observed in 2.3% of pts. Clinical activity was correlated with both dose and B7-H4 expression. For pts treated with doses ranging from 38.1–67.4 mg/m2 per cycle (intermediate dose range), the confirmed ORR in evaluable pts with high B7-H4 expression was 23% (6/26), including a 23% (3/13) confirmed ORR in evaluable pts with TNBC, with all 13 pts having previously received at least one topoisomerase-1 inhibitor (topo-1) ADC. At doses ≥76.2 mg/m2 per cycle (high dose range), the confirmed ORR in evaluable pts with high B7-H4 expression was 22% (2/9), with 78% (7/9) having ≥30% reduction in target lesions. Of the 8 pts with confirmed responses at doses ≥38.1 mg/m2, 5 had reduction in target lesions > 60%, including 1 CR. All 4 pts with high B7-H4 expression treated at the initial EXP dose of 67.4mg/m2 Q4W had tumor reductions and were on treatment with durations of ≥16 weeks as of data cutoff. Conclusions: Based on the initial reported data, Emi-Le appears to have encouraging clinical activity and tolerability in a heavily pretreated population. Further clinical development is ongoing in the EXP portion of the trial at a dose of 67.4 mg/m2 Q4W in pts with advanced/metastatic TNBC who have received 1-4 prior lines of systemic therapy, including at least one topo-1 ADC. Dose exploration is ongoing to identify a potential second higher EXP dose. Clinical trial information: NCT05377996 .

Background: Breast cancer (BC) treatment for trans feminine individuals is complicated by their use of gender-affirming estrogen therapy (ET). There is no clinical guideline to manage ET during BC treatment in these medically underserved patients. It is unknown whether continuing ET during BC treatment affects a successful outcome. We used a preclinical model to study the response of Brca1mut breast tumor to a PARP-inhibitor, in the presence of ET. Method: Tumor survival study using immune competent FVB/NJ mice was conducted by implanting fragments of K14-Cre Brca1f/fp53f/f tumors into female (n=45), male (n=28), and male mice receiving 60 µg of weekly subcutaneous ET (n=44). When the tumor reached 5 mm, male mice receiving ET were first randomized to stop or continue ET, then mice in each arm were randomized to be treated with a PARP inhibitor (olaparib) or were left untreated until study endpoint where the tumor reached 20 mm. A 10-day study in FVB/NJ mice was set up with the same eight arms as above (n=5/arm) whereby tumors were harvested after 10 days of olaparib treatment for RNASeq. Finally, to test the hypothesis that the male immune system modulated PARP inhibition, we conducted a six-arm tumor survival study in immune deficient NSG mice (n=15/arm): female+olaparib, female, male+olaparib, male, male+continued ET+olaparib, male+ continued ET. Log-rank test was used to compare tumor-specific survival. Differential gene expression was analyzed using limma. Gene set enrichment was conducted using mouse Hallmark gene sets and Camera. Results: Only arms treated with olaparib were reported here. In the tumor survival study using FVB/NJ mice, females had the longest median survival time of 103 days. Males had significantly a shorter survival than females (75 days, p=0.02). Males that stopped ET had significantly shorter survival (38.5 days) than females (p=0.003) and males (p=0.004). Males that continued ET also had shorter survival (50 days) than females (p=0.001) and males (p=0.16). There was no difference between males that stopped and continued ET (p=0.56). There was no survival difference between untreated study arms (median 14 to 19 days; p>0.05). No gene was differentially expressed between FVB/NJ male and female mice at FDR<0.05. Interferon alpha and gamma response gene sets were significantly upregulated in males compared to females while six gene sets (xenobiotic metabolism, bile acid metabolism, heme metabolism, reactive oxygen species, peroxisomes, and adipogenesis) were significantly downregulated (FDR<0.05). Tumors from males that continued ET were significantly upregulated for proliferative gene sets—G2M checkpoint, E2F targets, MYC targets, and mitotic spindle—compared to males, males that stopped ET, and females (FDR<0.05). Tumors from males that stopped ET showed decreased proliferation and increased inflammatory responses when compared to males, males that continued ET, and females (19 to 23 significantly enriched gene sets; FDR<0.05). The differences in tumor-specific survival previously observed in FVB/NJ mice were ablated in NSG mice. There was no difference between NSG females, males, and males that continued ET (median 69 days, all p>0.05). Conclusion: Male mice have poorer outcomes with PARP-inhibition than females, irrespective of cross-sex hormone treatment. Our survival study and transcriptomic data in immune competent mice suggested that although ET had profound molecular effects in the tumors, ET-modulated effects were not as strong as the effect of biological sex on olaparib efficacy. Our study in immune deficient mice confirmed that the male innate immune system played a major role in conferring poorer response to PARP-inhibition in males. Citation Format: Jan Heng, Lin Wang, Abhishek Thavamani, Erica S. Massicott, Brian R. Sardella, Vanessa C. Bret-Mounet, Zhaoji Liu, Steven R. Vandal, Emily K. Aronson, Gabrielle M. Baker, John G. Clohessy, Gerburg M. Wulf. Biological sex-linked immune modulation but not cross-sex estrogen therapy reduces the efficacy of PARP inhibition in the treatment of Brca1 breast tumors [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS12-08.

3028 Background: Cancer Associated Fibroblasts (CAFs) are key components of tumor microenvironment and have immunosuppressive functions. FAP is expressed in a restricted fashion on CAFs. OMTX705 is a first-in-class ADC targeting FAP with a novel tubulysin payload. OMTX705 demonstrated a good safety profile in relevant toxicology models and high linker stability in plasma. We report the dose escalation phase 1 trial of OMTX705 in monotherapy and in combination with pembrolizumab (PEM). Methods: Patients (pts) with advanced carcinomas or sarcomas received 1-18 mg/kg of OMTX705 monotherapy and 2-10 mg/kg with standard PEM. Escalation used a classical 3+3 design with backfilling. OMTX705 schedule is Day 1, 8 every 21 days. The primary endpoint is safety and key secondary are efficacy, pharmacokinetics and biomarkers. Biopsy and blood samples were collected for biomarker analysis. Results: A total of 78 pts have been dosed: 31 pts in monotherapy in 9 dose cohorts and 47 in combination in 7 cohorts of 3 pts each plus 2 backfilling cohorts at 4 and 7.5 mg/kg in pancreatic adenocarcinoma (PDAC) and microsatellite stable (MSS) colorectal cancer (CRC). Median age was 60, 43% male and ECOG PS 0 in 44%. Main histologies were PDAC 33% and MSS CRC 21% with median of 2 (1 to 5) and 3 (1 to 5) prior lines of therapy, respectively. Median treatment exposure was 44 days (range 8 to 113) in monotherapy and 92 days (1 to 422) in combination. OMTX705 relative dose intensity was ~100% in all dose levels. No DLT has been observed. The most frequent related TEAEs were asthenia 35%, AST increased 14%, diarrhea 8%, anemia 8%, and nausea 8%. Grade 3 related TEAEs (pts): anemia (2), immune-mediated hepatitis (2), GGT increased (1), neutropenia (1) and asthenia (1). In monotherapy, best response was SD in 26%. In combination, PR was achieved in 4% (1 MSS CRC and 1 PDAC; DOR 11+ and 8 months, respectively), SD in 33%, PD in 51%, and NE in 13%. In 13 pts there was target lesion reduction: median -17% (-46 to -1%). Median PFS was 1.4 months (0 to 14+). In combination, 20% PDAC (4/20) and 21% CRC (3/14) pts showed PFS > 4 months. 2/3 NSCLC with previous checkpoint inhibitor treatment, 2 PDAC, and 2 MSS CRC showed PFS > 7 months. High FAP expression (H-score > 30) was observed in 86% PDAC, 64% CRC and 50% other carcinomas. CD8+ and CD56+ immune-cell infiltration in tumor biopsies and downregulation of immunosuppressive cytokines in plasma samples were observed in PDAC and CRC best responders. OMTX705 tubulysin payload was detected in both CAFs and tumor epithelial apoptotic regions. Conclusions: OMTX705 is a novel anti-FAP ADC with excellent safety profile. The combination with PEM showed disease control in some heavily pretreated PDAC, MSS CRC and NSCLC. Changes in immune infiltrates and cytokines suggest that OMTX705 may revert CAF-mediated immunosuppression. Clinical trial information: NCT05547321 .

Mutations in BRCA1 and BRCA2 , whether inherited or somatically acquired, cause homologous recombination deficiency (HRD) in tumor cells. However, the precise timing of HRD onset in the tumor lineage is unknown. Under the haploinsufficiency model of BRCA1/2 , HRD-associated mutagenesis could begin as early as fertilization, whereas the prevailing two-hit tumor suppressor model places HRD onset upon bi-allelic loss, the exact timing of which remains undetermined. Here, we analyzed mutational signatures in 118 breast and ovarian cancer genomes exhibiting HRD to estimate the onset of HRD-driven mutagenesis prior to cancer diagnosis. Using HRDTimer, a novel algorithm, we estimate that HRD arises in tumor precursor cells at 37% of SBS1-based molecular time—corresponding to 8.6 years (IQR 7.8–9.7) prior to diagnosis in triple-negative breast cancers, and 16.0 years (IQR 14.2–18.5) in ER-positive breast cancers. Bulk sequencing reveals an accelerated accumulation of SBS1 mutations following neoplastic transformation, influencing the age estimate of HRD onset. Single-cell duplex sequencing confirms this pattern and further shows that nearly all non-tumor cells lack the HRD signature, indicating that HRD is rare in pre-malignant cells, even in patients with inherited BRCA1/2 mutations. Our analysis provides an estimate of the time window during which HRD mutagenesis emerges years before diagnosis, offering quantitative insight into when detection and potential interception may be possible. ### Competing Interest Statement The authors have declared no competing interest. Dana Farber/ Harvard Cancer Center Breast Cancer SPORE, 1P50CA168504-08 The Fund for Innovation in Cancer Informatics NIH - Somatic Mosaicism Across Human Tissues (SMaHT) grant

Combination chemotherapy remains essential for clinical management of triple-negative breast cancer (TNBC). Consequently, responses to multiple single agents cannot be delineated at the single patient level, even though some patients might not require all drugs in the combination. Herein, we conduct multi-omic analyses of orthotopic TNBC patient-derived xenografts (PDXs) treated with single agent carboplatin, docetaxel, or the combination. Combination responses were usually no better than the best single agent, with enhanced response in only ~13% of PDX, and apparent antagonism in a comparable percentage. Single-omic comparisons showed largely non-overlapping results between genes associated with single agent and combination treatments that could be validated in independent patient cohorts. Multi-omic analyses of PDXs identified agent-specific biomarkers/biomarker combinations, nominating high Cytokeratin-5 (KRT5) as a general marker of responsiveness. Notably, integrating proteomic with transcriptomic data improved predictive modeling of pathologic complete response to combination chemotherapy. PDXs refractory to all treatments were enriched for signatures of dysregulated mitochondrial function. Targeting this process indirectly in a PDX with HDAC inhibition plus chemotherapy in vivo overcomes chemoresistance. These results suggest possible resistance mechanisms and therapeutic strategies in TNBC to overcome chemoresistance, and potentially allow optimization of chemotherapeutic regimens.

Introduction: Triple-negative breast cancer (TNBC) patients frequently receive combination chemotherapy treatment, including most recently taxane/platinum combinations. However, treatment is not biomarker-guided. As such, it is not known which patient will respond to one or the other agent, or indeed which patients actually require the combination for the most effective treatment. We hypothesized that optimization of chemotherapy may be possible if molecular mechanisms and biomarkers underlying response to individual treatments can be identified. We evaluated this hypothesis in a preclinical trial using a cohort of 50 patient-derived xenograft (PDX) models of TNBC treated with either single-agent docetaxel or carboplatin, or their combination. Methods: 50 TNBC PDXs were evaluated for response to four weekly treatments with either single agent docetaxel (20 mg/kg), or carboplatin (50 mg/kg); 42 of these were also treated with their combination. Multi-omics profiling (genomics, transcriptomics, proteomics) was conducted before treatment. Gene level associations by treatment type were used to construct consensus gene sets by integrating our data with external TNBC PDX and patient cohorts which were then used to build XGBoost models to predict treatment-specific responses. Pathway analysis was performed using signed -log10 p-values from gene level results as input for Gene Set Enrichment Analysis. Results: Direct comparison of responses to carboplatin, docetaxel, and their combination showed that combination treatment was largely ineffective at generating enhanced responses over the best single agent. Only 13% of the 42 PDX showed enhanced responses in combination, with a comparable percentage (12%) showing antagonism between docetaxel and carboplatin, a phenomenon observed previously in vitro. Proteogenomic profiles revealed distinct genes associated with responses to each agent and their combination, suggesting different molecular mechanisms underlying each treatment response. A substantial number of genes linked to single-agent and combination treatments were validated in multiple independent PDX and patient cohorts receiving platinum and taxane-containing neoadjuvant therapy, confirming the clinical relevance of our PDX panel. Chemotherapy-specific predictors for pathological complete response (pCR)/CR achieved AUROCs of 0.79, 0.67, and 0.70 for platinum, taxane, and combination treatments, respectively. The single-agent platinum model was the most effective in predicting platinum response, with a similar trend for taxane and platinum+taxane predictors. These findings reinforce the observation that distinct gene sets are linked to responses to different chemotherapy treatments. Since the predictors used treatment-associated genes found in both PDX and clinical samples, these results suggest biomarker combinations for translation and clinical development to select TNBC tumors that may respond to specific regimens. In PDXs responsive to any treatment, several basal cytokeratins were among the top upregulated proteins compared to PDXs resistant to all treatments. KRT5 was validated by IHC in PDX tumors, achieving an AUROC of 0.83 for predicting responsiveness, indicating its potential as a future chemoresponse marker in TNBC. PDXs refractory to all treatment arms had higher levels of mitochondrial and cancer stem-cell-related pathways. Treatment with romidepsin, an HDAC inhibitor that targets these pathways and increases DNA damage, enhanced carboplatin response in a chemoresistant PDX model with high abundance of HDAC proteins. Conclusion: Proteogenomic characterization identifies molecular mechanisms and putative biomarkers for stratifying TNBC tumors for single or combination chemotherapy treatments, suggests targeted therapies to augment chemotherapy response, and provides a valuable resource for researchers and clinicians. Citation Format: Jonathan Lei, Lacey E. Dobrolecki, Chen Huang, Ramakrishnan R. Srinivasan, Suhas Vasaikar, Alaina N. Lewis, Christina Sallas, John D. Landua, Chang In Moon, Yuxing Liao, Na Zhao, Jin Cao, Susan G. Hilsenbeck, C. Kent Osborne, Mothaffar F. Rimawi, Matthew J. Ellis, Varduhi Petrosyan, Bo Wen, Kai Li, Alexander B. Saltzman, Antrix Jain, Anna Malovannaya, Gerburg Wulf, Shunqiang Li, Daniel C. Kraushaar, Elisabetta Marangoni, Tao Wang, Senthil Damodaran, Xiaofeng Zheng, Funda Meric-Bernstam, Bryan E. Welm, Alana L. Welm, Xi Chen, Gloria V. Echeverria, Meenakshi Anurag, Bing Zhang, Michael T. Lewis. Patient-derived Xenografts (PDX) Allow Deconvolution of Combination Chemotherapy Response [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS4-02.

Breast cancer is the most prevalent cancer among women worldwide with hormone receptor-positive (HR+) cancer constituting 70% of all breast cancers. HR+ breast cancer patients are usually treated with targeted therapies, which include hormone therapy, as well as CDK4/6- and PI3K-inhibitors. Unfortunately, the tumors usually become resistant over time. In addition to genetic drivers of resistance, metabolic perturbations and enhanced activity of metabolic enzymes can also contribute to tumor growth, resistance and poor survival. We have discovered that Fumarylacetoacetate hydrolase (FAH), the last enzyme in tyrosine catabolism pathway, is enriched in HR+ breast tumors and correlates with poor survival. By using HR+ patient-derived organoid cultures (PDOs), we show that FAH stimulates resistance to multiple standard-of-care therapies. Despite its canonical function in tyrosine catabolism, FAH overexpressing cells do not preferentially utilize the tyrosine catabolism pathway. Instead, we surprisingly observe increased nuclear localization of FAH in patients treated with endocrine therapy and/or CDK4/6-inhibitors. Similarly, ER degraders and CDK4/6-inhibitors increase the ratio of nuclear FAH in patient-derived organoids, suggesting that the nuclear localization could drive resistance. To investigate this, we cloned a nuclear localization signal (NLS) to FAH and observed that FAH-NLS expressing PDOs were more resistant to CDK4/6-inhibitors. In contrast, blocking the nuclear translocation reversed the FAH-mediated resistance, showing that the nuclear localization is essential for the resistance phenotype. Collectively this suggests a yet uncharacterized moonlighting function of FAH in the nucleus and links this metabolic enzyme to treatment resistance in HR+ breast cancer. Jenny Hogstrom, Boryana Petrova, Hanna Doh, Camila Perea, Su Min Hong, Nina Kozlova, Kayla Cruz, Conor L. Evans, Jonah Lee, John Clohessy, Gerburg Wulf, Laura Collins, Naama Kanarek, Taru Muranen. Fumarylacetoacetate hydrolase (FAH) stimulates resistance to CDK4/6 inhibitors in hormone receptor-positive breast cancer via a novel non-canonical function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3066.

TPS1141 Background: Breast cancer is the leading cause of cancer death for women worldwide, with triple-negative breast cancer (TNBC) considered one of the more aggressive breast cancers, accounting for ~15% of all cases. Unfortunately, there remains an unmet medical need for effective and well-tolerated treatments for advanced/metastatic TNBC; in heavily pretreated patients, standard-of-care single-agent chemotherapy has limited efficacy, with response rates of ~5%, PFS ~7 weeks. Emiltatug ledadotin (Emi-Le; XMT-1660) is a B7-H4-directed Dolasynthen ADC designed with a precise, target-optimized drug-to-antibody ratio (DAR 6) and a proprietary auristatin F-HPA microtubule inhibitor payload with controlled bystander effect. The FDA has granted Emi-Le two Fast Track designations for the treatment of adult patients with breast cancer, including patients with TNBC who have previously been treated with topoisomerase-1 inhibitor (topo-1) ADCs. Initial dose escalation clinical data from the ongoing Phase 1 trial at doses ranging from 38.1-67.4 mg/m2 per cycle demonstrated a 23% confirmed response rate in patients with B7-H4 high TNBC who were heavily pretreated all of whom received at least one prior topo-1 ADC. Methods: Based on encouraging clinical activity and tolerability data in the initial dose escalation data, the expansion portion (EXP) of the Phase 1 trial has been initiated and is actively enrolling patients. EXP has a Simon 2-stage design and will evaluate two doses in patients with advanced/metastatic TNBC who have received 1-4 prior lines of systemic therapy, including at least one topo-1 ADC. Patients will be evaluated for B7-H4 expression prospectively by IHC and will be stratified into B7-H4 TPS “high” and B7-H4 TPS “low” cohorts. The first EXP dose is 67.4 mg/m2 Q4W. Dose exploration is ongoing to identify a potential second higher EXP dose. The protocol includes the option for multiple additional indications, including HR+/HER2- breast cancer, endometrial cancer, ovarian cancer, and ACC-1. Clinical trial information: NCT05377996 .

BACKGROUND:CDK4/6 inhibitors (CDK4/6i) are used for management of hormone receptor-positive (HR+) metastatic breast cancer (MBC), and activation of the RAS/MAPK and PI3K/AKT signalling pathways has been implicated in resistance to these agents. Pathogenic NF1 mutations (pNF1m) dysregulate RAS signalling, but NF1 has not been linked to CDK4/6i resistance. We analysed multi-institutional data, real-world evidence, and preclinical models to characterise the impact of pNF1m on CDK4/6i sensitivity. METHODS:A retrospective cohort of patients with pNF1m tumours were identified from 4 institutions between 2/2015-5/2023 and evaluated for progression-free survival and intrinsic/acquired resistance on CDK4/6i. Real-world clinical-genomic data from GuardantINFORM between 6/2014 and 3/2023 was analysed for associations between pNF1m and time-to-next-treatment or overall survival following CDK4/6i, adjusted using propensity score weighting. We used CRISPR/Cas9 to delete NF1 in MCF7 and T47D breast cancer cells in vitro. NF1-knockout (NF1-KO) and -wild-type (WT) cells were analysed with respect to CDK4/6i sensitivity, MAPK and PI3K pathway activation, and sensitivity to MAPK and PI3K pathway inhibitors. In parallel, we assessed treatment response in a patient-derived organoid (PDO) harbouring NF1 loss, established from an HR+/HER2- breast tumor following progression on a CDK4/6i. FINDINGS:Among 1962 multicentre patients, we identified 38 with HR+/HER2- MBC, pNF1m, and exposure to CDK4/6i. NF1-associated intrinsic or acquired resistance to CDK4/6i was observed in a majority of tumours, and in those with baseline pNF1m on first-line CDK4/6i, a median progression-free survival of 6.2 months was much less than expected in routine practice. Real-world weighted analysis of 1161 patients comparing 28 pNF1m to 1133 NF1 non-altered tumours demonstrated shorter time-to-next-treatment on CDK4/6i regimens (4.2 vs. 12.4 months, hazard ratio 3.14, 95% confidence interval 2.01-4.93) and overall survival (15.8 vs. 45.2 months, hazard ratio 2.04, 95% confidence interval 1.09-3.82). NF1-deleted cells exhibited reduced sensitivity to CDK4/6i with or without oestrogen suppression, which was accompanied by induction of both MAPK and PI3K pathways, the latter of which was exacerbated by CDK4/6i. Blockade of RAS or AKT, but not MEK or ERK, reversed CDK4/6i resistance mediated by NF1 loss in cell lines and the PDO. INTERPRETATION:NF1 mutations are associated with shorter therapy duration on CDK4/6i in MBC. A causal link between NF1 loss and CDK4/6i resistance was supported by experiments in HR + breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, and blockade of RAS or AKT combined with CDK4/6i was effective in NF1-deleted cells and an NF1-mutant PDO. FUNDING:Breast Cancer Research Foundation DRC-20-001, National Cancer Institute R01CA273246, National Institute of Health P30 CA142543, Susan G. Komen Breast Cancer Foundation SAB1800010, Department of Defence BC 210406, Mary Kay Ash Foundation International Postdoctoral Scholars in Cancer Research Fellowship.

Significantly Altered Levels of Proteins Following Treatment with Copanlisib

Apoptotic induction following treatment with eribulin and copanlisib, either alone or in combination by IHC. Representative IHC pictures (A) and quantifications of IHC scores (B) of cleaved PARP on WHIM29 and WHIM34 PDX tumors harvested on day 3 following treatment with either vehicle (day 1), eribulin (day 1), copanlisib (days 1 and 2), or the combination, and FDG PET 4 hours after treatment on day 2.

BACKGROUND:The effect of gender-affirming testosterone therapy (TT) on breast cancer risk is unclear. This study investigated the association between TT and breast tissue composition and breast tissue density in trans masculine individuals (TMIs). METHODS:Of the 444 TMIs who underwent chest-contouring surgeries between 2013 and 2019, breast tissue composition was assessed in 425 TMIs by the pathologists (categories of lobular atrophy and stromal composition) and using our automated deep-learning algorithm (% epithelium, % fibrous stroma, and % fat). Forty-two out of 444 TMIs had mammography prior to surgery and their breast tissue density was read by a radiologist. Mammography digital files, available for 25/42 TMIs, were analyzed using the LIBRA software to obtain percent density, absolute dense area, and absolute non-dense area. Linear regression was used to describe the associations between duration of TT use and breast tissue composition or breast tissue density measures, while adjusting for potential confounders. Analyses stratified by body mass index were also conducted. RESULTS:Longer duration of TT use was associated with increasing degrees of lobular atrophy (p < 0.001) but not fibrous content (p = 0.82). Every 6 months of TT was associated with decreasing amounts of epithelium (exp(β) = 0.97, 95% CI 0.95,0.98, adj p = 0.005) and fibrous stroma (exp(β) = 0.99, 95% CI 0.98,1.00, adj p = 0.05), but not fat (exp(β) = 1.01, 95%CI 0.98,1.05, adj p = 0.39). The effect of TT on breast epithelium was attenuated in overweight/obese TMIs (exp(β) = 0.98, 95% CI 0.95,1.01, adj p = 0.14). When comparing TT users versus non-users, TT users had 28% less epithelium (exp(β) = 0.72, 95% CI 0.58,0.90, adj p = 0.003). There was no association between TT and radiologist's breast density assessment (p = 0.58) or LIBRA measurements (p > 0.05). CONCLUSIONS:TT decreases breast epithelium, but this effect is attenuated in overweight/obese TMIs. TT has the potential to affect the breast cancer risk of TMIs. Further studies are warranted to elucidate the effect of TT on breast density and breast cancer risk.

Supplementary Figure S1: PI3K pathway activity in selected cases with acquired PTEN alteration. Supplementary Figure S2. Validation of AKT constructs expression in T47D cells. Supplementary Figure S3. AKT activating mutations confer resistance to PI3Ka inhibitors. Supplementary Figure S4: Free energy calculations predict resistance to orthosteric PI3K inhibitors due to specific double PIK3CA mutants. Supplementary Figure S5: Free energy perturbation predicts reduced binding of orthosteric PI3K inhibitors to double mutants. Supplementary Figure S6. Expression of PIK3CA mutations in T47D cells. Supplementary Figure S7. MCF7 cells expressing W780R or Q859H double mutants show differential response to PIK3CA orthosteric inhibitors. Supplementary Figure S8: Chemical structure of RLY-2608. Supplementary Figure S9: Surface plasmon resonance (SPR) binding assay. Supplementary Figure S10. Alpelisib shows reduced potency of downstream signaling inhibition in the presence of W780R or Q859H/K. Supplementary Figure S11. T47D cells expressing I817F or E726K double mutants do not show a differential response to inavolisib (A) or RLY-2608 (B).