Triple Negative Breast Cancer Patient-derived Xenograft Models Research Articles

Targeting neddylation and sumoylation in chemoresistant triple negative breast cancer

Triple negative breast cancer (TNBC) accounts for 15–20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.

Evaluation of copanlisib in combination with eribulin in triple negative breast cancer patient-derived xenograft models.

The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-29-01.

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Abstract PO2-28-08: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract TNBC is one of the most aggressive subtypes of breast cancer. Combating chemotherapy resistance is critical to improving quality of care and reducing fatality among TNBC patients. In order to understand the mechanisms of chemoresistance in TNBC tumors, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy among tumors, and metastatic potential of certain tumors over others. Thus, this study utilizes spatially resolved transcriptomic (SRT) technology to profile the spatial interactions in the TNBC TME. With the goal of better understanding chemoresistance in TNBC and providing a proof-of-concept for new technology, we have launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Genomics Visium was performed on vehicle, AC-treated residual tumor (21 days post-Tx), and AC-treated regrown tumor (50 days post-Tx when tumors regrew to starting tumor volume). This study is divided into two parts: 1) development of a computational pipeline; 2) spatial colocalization analysis of residual and regrown tumors to understand chemoresistance. PART 1: Due to the lack of specialized tools for processing and sorting xenograft reads from SRT data, we developed the Xenomake pipeline, which combines a xenograft sorting algorithm (Xengsort) and spatial barcode demultiplexing pipeline to assign reads into the host and graft organisms for each spatial spot. RESULTS: Xenomake permits clustering the spatial spots into stroma-rich (enriched for mouse mRNAs), and epithelial-rich (enriched for human mRNAs) regions. We show that Xenomake can find differential cytokine production in the stroma and epithelium. Since PDX data separate the tumor into stroma and epithelium by organism, the pipeline enables fine-tuned downstream analysis such as stromal-stromal and stromal-epithelial interactions. Xenomake is thus generally applicable for SRT involving PDX samples. PART 2: Previously we detailed patterns of tumor regression into a residual tumor state, followed by uncontrolled regrowth in the absence of treatment in multiple PDX models of TNBC. Although using PDX models necessitates immune-compromised mice, several types of stromal populations are present and analyzable in these models. Thus, using organism-assigned reads processed by Xenomake, we computationally inferred the localization pattern of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial cells (ENDO), monocytes, and perivascular-like cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for the ~30 HALLMARK pathways across samples. We correlated the patterns of SPAM with stroma cell-type SLP to survey the stroma-epithelial interactions across samples. RESULTS: Stroma cells are generally distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and with CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Furthermore, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-08.

Abstract PO1-06-14: Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC)

Abstract Background: Aberrant PI3K pathway signaling is frequently observed in TNBC. Increasing evidence indicate that PI3K pathway activation maintains the stemness and chemoresistance of breast cancer stem cells (CSCs). PI3K inhibition sensitizes CSCs to chemotherapy. Eribulin (E), a non-taxane microtubule dynamics inhibitor, showed overall survival benefit in metastatic HER2 negative BC. Preclinically, the addition of copanlisib (C), a potent pan-class I PI3K inhibitor that is highly selective against α and δ PI3K isoforms, improved anti-tumor effect in both E-sensitive and resistant TNBC patient-derived xenograft models, irrespective of PIK3CA/PTEN alteration status. Here we report the preliminary data of the phase I dose escalation trial of E+C in patients with metastatic TNBC. Methods: This trial includes a phase I study to determine the dose limiting toxicity (DLT) and recommended phase 2 dose (RP2D) of E+C, followed by a randomized phase II study of E+C (at RP2D) versus E (stratified by tumor PIK3CA/PTEN mutation status) with progression-free survival (PFS) as the primary endpoint. Key eligibility included patients with: metastatic TNBC who progressed on ≤5 lines of chemotherapy, prior anthracycline/taxane chemotherapy (unless contraindicated), ECOG 0-1, adequate organ function and known tumor PIK3CA/PTEN/AKT mutation status. Key exclusions include: prior receipt of E or any PI3K/mTOR/AKT inhibitor, grade ≥2 neuropathy, presence of tumor AKT mutation, and uncontrolled diabetes, hypertension or congenital QT prolongation. A 3+3 design was used to determine the DLT and RP2D. Dose level 1 (DL1) started with E at 1.1 mg/m2 mg IV and C at 45 mg IV on days (D) 1/8 of 21-D cycle, with the potential to escalate to 2 additional dose levels – DL2: E 1.4 mg/m2 and C 45 mg on D 1/8; DL3: E 1.4 mg/m2 and C 60 mg on D 1/8. Results: Eight patients were enrolled in the phase I study and received at least 1 dose of E+C. The median age was 48.5 (range, 26-67) years. The majority of patients identified as White (additionally 2 Hispanic, 1 Black) and had an ECOG performance status of 1 (n=6, 75.0%). Tumor PIK3CA and PTEN alterations were each observed in 1 patient. Median number of cycles received were 2 (range, 1-5). All 8 patients had at least 1 treatment-related adverse event (TRAE). The most common TRAEs across all dose levels were: lymphopenia (n=5, 62.5%), neutropenia (n=5, 62.5%), hyperglycemia (n=4, 50.0%), anemia (n=4, 50.0%) and fatigue (n=4, 50.0%); most toxicities were grade (G) 1-2. G3 hyperglycemia and hypertension were observed in 1 patient each, were medically managed and did not lead to treatment interruption. 1 patient was diagnosed with pulmonary embolism, possibly attributed to study therapy and breast cancer. DLTs were reported in 2 of 2 patients treated at DL2 - 1 patient developed G3 rash responsive to oral corticosteroids, and 1 patient developed G3 acute kidney injury in the setting of hypovolemia and discontinued study therapy. No additional patients were enrolled in DL2. No DLTs were observed in all 6 patients treated at DL1. 1 patient treated at DL1 had possible G1 non-infectious pneumonitis that resolved spontaneously on short interval imaging. Stable disease was observed in 2 of 6 response-evaluable patients (33.3%). 1 patient achieved regression of cutaneous disease following 1 cycle of therapy as assessed by the treating investigator. DL1, E at 1.1 mg/m2 plus C at 45 mg IV, was determined to be the RP2D. Tumor tissue biomarker analysis is underway. Conclusion: These data indicate that E+C at DL1 is a well-tolerated regimen warranting further investigation. The study is actively enrolling patients to the phase II study. Clinical trial information: NCT04345913. Funding Source: National Cancer Institute. Citation Format: Nusayba Bagegni, Brittney Haas, Leslie Nehring, Jingqin Luo, Laura Kennedy, Meghna Trivedi, Manali Bhave, Rabih Said, Cynthia Ma. Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-14.

Abstract 1986: Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts

Abstract Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1986.

Abstract 6620: PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease

Abstract Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6620.

Abstract 6429: Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer

Abstract Objective: In the cancer research, a highly promising in vivo platform for personalized treatment involves the adoption of an advanced approach—specifically, the grafting of patient-derived xenografts (PDX) onto humanized mouse models. These models capture distinctive aspects of interaction with the human immune system and the tumor microenvironment (TME). In this study, we have engineered humanized triple-negative breast cancer (TNBC) PDX models to observe alterations in differentially expressed genes, thereby enhancing our understanding of the intricacies of personalized treatment in this context. Method: We utilized two TNBC PDX models established for the humanized models, where CD34+ hematopoietic stem cells were transplanted into immunodeficient mice. RNA sequencing was performed to identify altered gene expression in humanized PDX models compared to NSG PDXs. Additionally, we generated stable cell lines overexpressing TMEM176B in MDAMB231 and Hs578T cells, and we knocked down TMEM176B in 4T1 cells using recombinant techniques Results: In the humanized model, the growth of PDX tumors did not show a significantly faster or markedly different rate compared to previous studies. However, upon conducting RNA sequencing in the PDX model, the expression of the TMEM176B gene was consistently downregulated. Consequently, we transfected and observed that the increased expression of TMEM176B in MDAMB231 and Hs578T cell lines resulted in enhanced cell proliferation in vitro. Transplanting MDAMB231 cells overexpressing TMEM176B into athymic nude mice resulted in accelerated tumor growth. The study further investigates the expression of TMEM176B in an immune-competent environment by knocking down the gene in 4T1 cells or transplanting the control group into a balb/c mouse model. Tumors with TMEM176B knockdown exhibited inhibited growth, increased CD3+ T cell infiltration, and significant inhibition when treated with anti-PD-L1 compared to the control group. Conclusion: This research demonstrates the utility of PDX models, particularly humanized PDX models, in studying the complexities of cancer biology and the tumor microenvironment. The findings regarding TMEM176B and its role in tumorigenesis and immune responses in triple-negative breast cancer provide valuable insights for further research and potential therapeutic interventions. Citation Format: Yujeong Her, Jihui Yun, Woohang Heo, Jong-Il Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6429.

Potential Perturbations of Critical Cancer-regulatory Genes in Triple-Negative Breast Cancer Cells Within the Humanized Microenvironment of Patient-derived Xenograft Models.

In this study, we aimed to establish humanized patient-derived xenograft (PDX) models for triple-negative breast cancer (TNBC) using cord blood (CB) hematopoietic stem cells (HSCs). Additionally, we attempted to characterize the immune microenvironment of the humanized PDX model to understand the potential implications of altered tumor-immune interactions in the humanized PDX model on the behavior of TNBC cells. To establish a humanized mouse model, high-purity CD34+ HSCs from CB were transplanted into immunodeficient NOD scid γ mice. Peripheral and intratumoral immune cell compositions of humanized and non-humanized mice were compared. Additionally, RNA sequencing of the tumor tissues was performed to characterize the gene expression features associated with humanization. After transplanting the CD34+ HSCs, CD45+ human immune cells appeared within five weeks. A humanized mouse model showed viable human immune cells in the peripheral blood, lymphoid organs, and in the tumor microenvironment. Humanized TNBC PDX models showed varying rates of tumor growth compared to that of non-humanized mice. RNA sequencing of the tumor tissue showed significant alterations in tumor tissues from the humanized models. tumor necrosis factor receptor superfamily member 11B (TNFRSF11B) is a shared downregulated gene in tumor tissues from humanized models. Silencing of TNFRSF11B in TNBC cell lines significantly reduced cell proliferation, migration, and invasion in vitro. Additionally, TNFRSF11B silenced cells showed decreased tumorigenicity and metastatic capacity in vivo. Humanized PDX models successfully recreated tumor-immune interactions in TNBC. TNFRSF11B, a commonly downregulated gene in humanized PDX models, may play a key role in tumor growth and metastasis. Differential tumor growth rates and gene expression patterns highlighted the complexities of the immune response in the tumor microenvironment of humanized PDX models.

Abstract LB_C04: Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models

Abstract Background: Cyclin/cyclin-dependent kinase (CDK) complexes regulate the activity of RB1 and E2F to drive cell cycle progression while ensuring fidelity of DNA replication. The interaction between cyclins and a critical subset of their substrates, including RB1 and E2F, is mediated by a conserved short linear RxL motif. RxL peptides have been shown to inhibit substrate phosphorylation by CDK2/cyclin A (AACR 2022 poster #5379). Previously, we have shown that macrocycles that selectively inhibit RxL-mediated binding to cyclins A and B (RxL inhibitors) lead to growth inhibitory activity in multiple cancer cell lines, and result in tumor regression in small cell lung cancer (SCLC) and ovarian cancer xenograft models (AACR 2023 poster #1560). Here, we test the antitumor activity of CID-016 in patient-derived xenograft (PDX) models from breast cancer. Methods: Bioinformatic analyses using RNA sequencing (RNAseq) and whole exome sequencing (WES) data from 39 breast cancer cell lines was employed to identify biomarker profiles that associate with sensitivity to CID-016. The antiproliferative activity of CID-016 was assessed by GI50 after 4-8 days of treatment. Hallmark pathway scores were calculated by Gene Set Variation Analysis (GSVA) method using MSigDb Hallmark collection from cell lines and existing PDX models. Based on the calculated hallmark pathway score, triple negative breast cancer (TNBC) PDX models were selected to test the anti-tumor activity of CID-016. In vivo, NMRI-Foxn1nu/nu mice were implanted with tumor fragments in the lower flank and treated with vehicle or CID-016 oral formulation of 50-100 mg/kg BID or 100 mg/kg QD, respectively for at least 28 days. Tumor volume and body weight were measured biweekly. Pharmacodynamic (PD) analyses will be conducted after treatment with CID-016. Results: Treatment of the breast cancer cell line panel with CID-016 demonstrated greater potency by GI50 in TNBC compared to other breast cancer cell lines (p=0.009). Gene expression levels of CCNB1, CCNA2, CCNE1 as well as target hallmark scores for “E2F targets”, “G2M checkpoint” and “Mitotic spindle” were significantly higher in TNBC vs other breast cancer cell lines. Oral treatment with CID-016 resulted in tumor regression in at least one triple-negative breast cancer PDX model and was well tolerated with no significant body weight changes. In vivo PD analyses will be assessed after 5 days of treatment with CID-016. Conclusions: The selective cyclin A/B RxL inhibitor CID-016 exhibits potent antitumor activity in TNBC preclinical models, consistent with the proposed mechanism of RB1/E2F pathway dysregulation. Given their compelling characteristics, orally bioavailable RxL inhibitors are advancing as the only first-in-class orally bioavailable dual inhibitors of cyclins A and B into clinical development. Citation Format: Mariana Paes Dias, Li-Fen Liu, Bernard Levin, Cristina Molina, Marta Guzmán, Olga Rodríguez, Evelyn W. Wang, Violeta Serra. Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C04.

Discovery of VIP236, an αvβ3-Targeted Small-Molecule-Drug Conjugate with Neutrophil Elastase-Mediated Activation of 7-Ethyl Camptothecin Payload for Treatment of Solid Tumors.

The emerging field of small-molecule-drug conjugates (SMDCs) using small-molecule biomarker-targeted compounds for tumor homing may provide new perspectives for targeted delivery. Here, for the first time, we disclose the structure and the synthesis of VIP236, an SMDC designed for the treatment of metastatic solid tumors by targeting αvβ3 integrins and extracellular cleavage of the 7-ethyl camptothecin payload by neutrophil elastase in the tumor microenvironment. Imaging studies in the Lewis lung mouse model using an elastase cleavable quenched substrate showed pronounced elastase activity in the tumor. Pharmacokinetics studies of VIP236 in tumor-bearing mice demonstrated high stability of the SMDC in plasma and high tumor accumulation of the cleaved payload. Studies in bile-duct-cannulated rats showed that biliary excretion of the unmodified conjugate is the primary route of elimination. Treatment- and time-dependent phosphorylation of H2AX, a marker of DNA damage downstream of topoisomerase 1 inhibition, verified the on-target activity of the payload cleaved from VIP236 in vivo. Treatment with VIP236 resulted in long-lasting tumor regression in subcutaneous patient-derived xenograft (PDX) models from patients with non-small-cell lung, colon, and renal cancer as well as in two orthotopic metastatic triple-negative breast cancer PDX models. In these models, a significant reduction of brain and lung metastases also was observed.

Abstract 6553: The development of homologous recombination repair deficiency (HRD) score methodology using WES data and its application in patient-derived xenograft models

Abstract Homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRR) are associated with sensitivity to platinum-based chemotherapy, topoisomerase 1 inhibitors and inhibitors targeting DNA repair pathway. Use of the Myriad HRD test for HRD determination in preclinical models is not feasible globally. To address this challenge, we developed an approach to generate HRD scores from whole exome sequencing (WES) data and applied it to assess HRD status in patient-derived xenograft (PDX) models. We used python package CNVkit for segmentation and gene-level/allele-specific copy number calling with customized settings according to CNVkit documentation. The reference was constructed using the WES BED file and hg38 reference genome when there are no paired control samples available. Next R package scarHRD was used to determine the HRD score that is the sum of loss of heterozygosity (LOH), telomere allele imbalances (TAI), and large fragment migrations (LST). The method was applied to the WES of 25 Xentech triple negative breast cancer (TNBC) PDX models to derive HRD scores. The samples were also separately profiled using Myriad’s MyChoice CDx assay. The two scores showed moderate correlation with Pearson r = 0.56. However, when n = 6 PDX models with low coverages (average depth <100x) were excluded, the Pearson r improved to r = 0.76. We also found that the absolute value of HRD score calculated by CNVkit/scarHRD is lower than that of Myriad HRD score. Furthermore, we evaluated the genomic landscape in this cohort of TNBC PDX models for the selected genes of HRR pathway (BRCA1, BRCA2, ATM, BRIP1, PALB2, RAD51C, BARD1, CDK12, CHEK1/2, FANCL, RAD51B, RAD51D, RAD54L). Sixteen models found to carry HRR mutations, with ATM and BRCA1 mutations being the most common ones, being found in 9 and 5 of 25 models, respectively. Although HRRm positive (HRRm+) models have higher HRD score for both Myriad HRD score and HRD score generated with CNVkit/scarHRD approach, the association between HRR (HRRm+/HRRm-) and Myriad HRD (HRD+/HRD- defined by HRD threshold of 42) is not statistically significant (Fisher’s exact test p value = 0.1998, odds ratio=3.5), likely due to small cohort sizes. These results suggest that combination of CNVkit and scarHRD for HRD score calculation may be an alternative approach for HRD identification in preclinical PDX models. Citation Format: Jixin Wang, Krista Kinneer, Wenyan Zhong, Zhongwu Lai. The development of homologous recombination repair deficiency (HRD) score methodology using WES data and its application in patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6553.

Discovery of a first-in-class ANXA3 degrader for the treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a nasty disease with extremely high malignancy and poor prognosis. Annexin A3 (ANXA3) is a potential prognosis biomarker, displaying an excellent correlation of ANXA3 overexpression with patients' poor prognosis. Silencing the expression of ANXA3 effectively inhibits the proliferation and metastasis of TNBC, suggesting that ANXA3 can be a promising therapeutic target to treat TNBC. Herein, we report a first-in-class ANXA3-targeted small molecule (R)-SL18, which demonstrated excellent anti-proliferative and anti-invasive activities to TNBC cells. (R)-SL18 directly bound to ANXA3 and increased its ubiquitination, thereby inducing ANXA3 degradation with moderate family selectivity. Importantly, (R)-SL18 showed a safe and effective therapeutic potency in a high ANXA3-expressing TNBC patient-derived xenograft model. Furthermore, (R)-SL18 could reduce the β-catenin level, and accordingly inhibit the Wnt/β-catenin signaling pathway in TNBC cells. Collectively, our data suggested that targeting degradation of ANXA3 by (R)-SL18 possesses the potential to treat TNBC.

Abstract P6-11-10: Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer

Abstract BACKGROUND: Nearly 50% of patients with triple negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NACT) retain residual tumors resulting in high rates of metastatic relapse and poor overall survival. Residual tumors surviving NACT (Adriamycin plus cyclophosphamide; AC) were found to undergo a metabolic transition to heightened mitochondrial oxidative phosphorylation (oxphos; PMID: 30996079). Pharmacologic inhibition of mitochondrial electron transport chain (ETC) complex I with IACS-010759 (PMID: 29892070) had enhanced efficacy in residual, rather than treatment-naïve, tumors of orthotopic patient-derived xenograft (PDX) models. Our analyses of mitochondrial structure and function in human TNBC cell lines revealed differing adaptations in residual cells surviving treatment with conventional NACT agents. While DNA-damaging chemotherapies (e.g.Adriamycin, carboplatin) induced mitochondrial fusion and oxphos, taxanes (e.g.paclitaxel, docetaxel) induced mitochondrial fragmentation and reduced oxphos (Baek et al., Biorxiv Doi 10.1101/2022.02.25.481996). The mechanistic basis of these mitochondrial adaptations is not yet understood. The mitochondrial ETC consists of 92 proteins, 13 of which are encoded in the mitochondrial genome (mtDNA) and translated by the mitoribosome, while the remaining are encoded by the nuclear genome (nDNA), translated by the cytoribosome, and inserted into the inner mitochondrial membrane by accessory proteins, namely Oxidase (Cytochrome C) Assembly 1-Like (OXA1L). Disruption of OXA1L in mammalian cells has been shown to affect the levels and activity of ETC complexes I, III, IV, and V, and thus diminish oxphos. We aim to determine whether mitochondrial translation and OXA1L activity represent therapeutic vulnerabilities to overcome pro-survival metabolic adaptations in chemoresistant TNBC thereby augmenting treatment response. METHODS: Weare evaluating the effects of conventional TNBC chemotherapies singly, and in standard combinations, on mitochondrial translation and ETC formation in human TNBC cells and PDX models(PIM001-P, WHIM14, BCM15116) using metabolomic and proteomic profiling. To perturb these processes genetically, we knocked down (KD) OXA1Lwith siRNA. We are complementing these studies pharmacologically using conventional antibiotics, such as tigecycline, as previous studies showed they inhibit mitochondrial translation in breast and other cancers (PMID: 25625193). These studies will reveal whether OXA1L and mitochondrial translation are required for metabolic adaption and chemotherapy resistance of residual TNBC cells. PDX preclinical trials based on our published residual tumor testing schema (PMID: 30996079), will reveal whether the sequential combination of NACT followed by tigecycline can effectively perturb residual tumor relapse. RESULTS: Proteomic profiling of longitudinally harvested PDX tumors demonstrates substantial disruption of mitochondria-and nuclear-encoded ETC components in residual vs. treatment-naïve tumors. Interestingly, these patterns are distinct between different chemotherapy treatments, with an increase of ETC components in carboplatin-treated residual tumors compared to a decrease in docetaxel-treated residual tumors. Western blot analyses of human cell lines show OXA1LKD perturbs levels of both nuclear-and mitochondria-encoded ETC components. Preliminary findings suggest OXA1LKD increases sensitivity to chemotherapies in human TNBC cell lines. Finally, tigecycline effectively inhibits TNBC cell growth. We next will evaluate whether residual cells not killed by conventional chemotherapies have enhanced tigecycline susceptibility. CONCLUSION: These data suggest targeting mitochondrial translation may be a promising approach to overcome pro-survival metabolic adaptations in residual TNBC cells not killed by conventional chemotherapies. Citation Format: Mariah J. Berner, Lily Baek, Junegoo Lee, Philip L. Lorenzi, Mei Leng, Alexander B. Saltzman, Anna Malovannaya, Lacey E. Dobrolecki, Christina Sallas, Michael T. Lewis, Gloria V. Echeverria. Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-10.

Abstract PD5-01: PD5-01 Genomic and epigenomic BRCA alterations predict adaptive resistance and response to platinum-based therapy in triple negative breast cancer and ovarian carcinoma

Abstract Background. Homologous recombination deficiency (HRD), induced by germline and somatic BRCA1 or BRCA2 gene mutations (BRCAmut) and by BRCA1 promoter methylation (BRCA1meth), has been associated with better response to platinum agents in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent studies is why patients with BRCA1meth cancers do more poorly compared to those with BRCAmut cancers given the biologically equivalent HRD in both states. Here, we address this question by performing detailed genomic analyses of primary TNBC and OvCa cohorts and through patient-derived xenografts (PDXs) and their derivative cell lines. Methods. Both new and publicly available cohorts of primary TNBC and OvCa encompassing 499 individuals treated with a combination of platinum and taxane chemotherapy were analyzed by whole genome and transcriptome sequencing as well as limited epigenetic analyses. A cohort of 43 PDX models of TNBC was genomically characterized and responses to both single agent platinum and docetaxel were evaluated in vivo. PDX longitudinal studies were performed to assess the dynamics of BRCA1 methylation following treatment. Results. Genomic analyses revealed that BRCA1mut and BRCA1meth cancers share the same pattern of BRCA1-linked genomic rearrangements. Nonetheless, in all four primary cancer cohorts examined we found that patients with BRCAmut cancers, but not those with BRCA1meth cancers, had significantly better response outcomes compared to those with BRCA proficient cancers. A separate analysis of PDX TNBCs with BRCA1 promoter methylation showed that PDXs derived from treatment naïve cancers had complete methylation of the BRCA1 promoter, whereas those derived from post-treatment cancers invariably had only partial methylation. Compared to PDXs with complete methylation, those with partial methylation had a lower response rate to in vivo platinum-based therapy, but not to docetaxel. Using single cell clonal expansions from BRCA1meth PDX models, we confirmed that partial methylation was the result of demethylation of one of the BRCA1 promoter alleles and not of the outgrowth of a non-methylated clone. Exposure of TNBC PDXs with complete methylation to a single course of platinum therapy resulted in the emergence of an unmethylated BRCA1 promoter allele, which associated with an increase in BRCA1 expression. We confirmed that platinum treatment results in progressive loss of BRCA1 methylation and restoration of BRCA1 expression in the clinical setting, by studying the BRCA1 status of a longitudinal series of four TNBC PDX models established from the same patient at different stages of her clinical history. Differential gene expression analysis revealed an increased immune transcriptional signal, especially an elevated M1 macrophage signature, associated with enhanced response to platinum therapy only in patients with BRCA proficient cancers, in both TNBC and OvCa. Integrating both the strength of this cancer immune signature and the presence of BRCA mutations resulted in more accurate predictions of response when compared to either HRD or BRCA status alone. Conclusions. These results suggest that unlike BRCAmut cancers, where BRCA deficiency is more genetically stable, BRCA1meth cancers are highly adaptive to genotoxin exposure resulting in demethylation of one allele, recovery of BRCA1 expression and acquired insensitivity to platinum. On the other end, a high immune transcriptional signature identifies patients with BRCA proficient cancers that are more likely to benefit from platinum therapy. Taken together, our study underscores the importance of characterizing molecular heterogeneity to optimize predictive precision in assigning response probabilities in TNBC and OvCa. Citation Format: Francesca Menghi, Kalyan Banda, Pooja Kumar, Robert Straub, Lacey E. Dobrolecki, Isabel Rodriguez, Susan E. Yost, Harshpreet Chandok, Marc Radke, George Somlo, Yuan Yuan, Michael T. Lewis, Elizabeth Swisher, Edison Liu. PD5-01 Genomic and epigenomic BRCA alterations predict adaptive resistance and response to platinum-based therapy in triple negative breast cancer and ovarian carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-01.

Predictors of success in establishing orthotopic patient-derived xenograft models of triple negative breast cancer

Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient’s diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient’s tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.

Phase 1 study of SGN-B7H4V, a novel, investigational vedotin antibody–drug conjugate directed to B7-H4, in patients with advanced solid tumors (SGNB7H4V-001, trial in progress).

TPS3155 Background: B7-H4, a B7 immune checkpoint ligand, is expressed at low levels in normal tissue and negatively regulates T-cell function by inhibiting T-cell proliferation and cytokine production. B7-H4 expression is elevated in solid tumors, including breast, ovarian, and endometrial cancers. Targeting B7-H4–expressing tumor cells may relieve B7-H4–mediated T-cell inhibition. SGN-B7H4V is a novel, investigational vedotin antibody–drug conjugate directed to B7-H4 with proposed mechanisms of action including monomethyl auristatin E (MMAE)-directed cytotoxicity, bystander effect, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis. SGN-B7H4V elicited antitumor activity in cell line-derived xenograft models of triple-negative breast cancer (TNBC) and patient-derived xenograft models of TNBC and ovarian cancer (Gray et al 2021), providing a rationale to evaluate SGN-B7H4V in patients (pts) with advanced solid tumors. Methods: SGNB7H4V-001 (NCT05194072) is a phase 1, first-in-human, multicenter, open-label trial evaluating the safety, tolerability, pharmacokinetics (PK), and antitumor activity of SGN-B7H4V in pts with advanced solid tumors. This study includes 3 parts: dose escalation (Part A), dose and schedule optimization (Part B), and dose expansion in disease-specific cohorts and a biology cohort (Part C). Adult pts (≥18 years) with histologically/cytologically confirmed locally advanced unresectable or metastatic solid tumors including high-grade serous epithelial ovarian cancer, primary peritoneal cancer, fallopian tube cancer, human epidermal growth factor receptor 2-negative and hormone receptor-positive breast cancer, TNBC, endometrial carcinoma, squamous non-small cell lung cancer, cholangiocarcinoma, or gallbladder carcinoma, are eligible. Pts must have ECOG PS 0–1 and relapsed/refractory disease or be intolerant to standard-of-care therapies. Prior treatment with an MMAE-containing agent or a B7-H4–targeted agent is not permitted. Primary endpoints include the rate of adverse events, laboratory abnormalities, dose-limiting toxicities, and cumulative dose-level safety. Secondary endpoints are objective response rate (ORR), complete response rate, duration of objective response (DOR), progression-free survival (PFS), overall survival (OS), PK, and incidence of antidrug antibodies. Exploratory endpoints include pharmacodynamic (PD) analyses, PD and PK exposure measurements, B7-H4 characterization on malignant cells, and biomarker analyses. Safety and antitumor activity endpoints will be assessed using descriptive statistics. ORR will be analyzed by tumor type and dose. DOR, PFS, and OS will be estimated using the Kaplan–Meier method. Enrollment for Part A is ongoing in North America and is planned in Europe. Clinical trial information: NCT05194072.

A phase I/II trial evaluating the safety and efficacy of eribulin in combination with copanlisib in patients with metastatic triple-negative breast cancer (TNBC).

TPS1128 Background: Metastatic (met) TNBC remains a clinical challenge with limited treatment options and inevitable chemoresistance. Aberrant PI3K pathway signaling is frequently observed in TNBC. Increasing evidence shows PI3K pathway activation maintains the stemness and chemoresistance of BC stem cells (CSCs), and PI3K inhibition sensitizes CSCs to chemotherapy (chemo). Eribulin (E), a non-taxane microtubule dynamics inhibitor, showed survival benefit in met HER2 negative BC. Preclinically, E impacts tumor vascular remodeling, inhibits epithelial-to-mesenchymal transition and metastasis – key mechanisms implicated in PI3K inhibition resistance. Copanlisib (C), a potent pan-class I PI3K inhibitor ( i), improved anti-tumor effect in E-sensitive and resistant TNBC patient-derived xenograft models, irrespective of PIK3CA/PTEN mutation (mut) status, when combined with E. This phase I/II study is aimed to determine the safety and efficacy of E+C in pts with met TNBC. Methods: This trial includes a phase I portion with the primary objective to determine the dose limiting toxicity (DLT) and recommended phase 2 dose (RP2D) of E+C, followed by a phase II randomized portion of E+C (at RP2D) versus ( vs) E with the primary objective of progression-free survival (PFS). Key secondary objectives include objective response rate (ORR) and clinical benefit rate (CBR) [phase I]; and ORR and CBR, by arm and by PIK3CA/PTEN mut status and assessment of treatment induced target engagement [phase II]. Key exploratory objectives include analysis of genomic, proteomic and metabolomic changes as potential response biomarkers in tumor tissue and blood. Key eligibility criteria include pts with: met TNBC who progressed on ≤5 chemo lines, including anthracycline/taxane (unless contraindicated), ECOG 0-1, adequate organ function and known archival tumor PIK3CA/PTEN mut status. Key exclusions: prior E or PI3K/mTOR/AKT i, grade ≥2 neuropathy, tumor AKT mut, congenital QT prolongation, and uncontrolled diabetes or hypertension. Phase I portion will follow a 3+3 design for E+C dose escalation to enroll 18 max pts, starting at E 1.1 mg/m2 IV and C 45 mg IV on days (D) 1/8 of 21-D cycle (C) (to E 1.4 mg/m2 and C 60 mg max). RP2D will be defined as the highest dose level at which at most 1 of 6 pts experience DLT during C1. 88 pts will be randomized (1:1) in the phase II portion to E+C vs E (1.4 mg/m2 D 1/8), stratified by PTEN/PIK3CA mut status. Response assessment by Response Evaluation Criteria in solid tumors (RECIST) v1.1 will occur every 9 weeks (+/-7 D). Tumor biopsy is required at baseline and C2D1-2, and optional at progression. A sample size of 88 achieves 80% power to detect PFS difference of median PFS 6.95 vs 4 months (corresponding to a hazard ratio of 0.5755) between the 2 arms, based on 1-sided two-sample log rank test at 0.1 α level. The phase I study is actively enrolling pts. Clinical trial information: NCT04345913.

Docosahexaenoic acid enrichment of tumor phospholipid membranes increases tumor necroptosis in mice bearing triple negative breast cancer patient-derived xenografts

Docosahexaenoic acid (DHA) reduces breast cancer tumor growth in preclinical models. To better understand how DHA amplifies the actions of docetaxel (TXT) chemotherapy, we examined the effects of two doses of dietary DHA on tumor size, membrane DHA content and necroptosis using a drug resistant triple negative breast cancer (TNBC) patient derived xenograft (PDX) model. Female NOD.Cb-PrkdcscidIl2rg mice bearing TNBC PDXs were randomized to one of three nutritionally complete diets (20% w/w fat): control (0% DHA), high DHA (3.8% HDHA), or low DHA (1.6% LDHA) with or without intraperitoneal injections of 5 mg/kg TXT, twice weekly for 6 weeks (n=8 per group). Tumors from mice fed either HDHA+TXT or LDHA+TXT were similar in size to each other, but were 36% and 32% smaller than tumors from mice fed control+TXT, respectively (P<.05). A dose effect of DHA incorporation was observed in plasma total phospholipids and in phosphatidylethanolamine and phosphatidylinositol. Both doses of DHA resulted in similarly increased necrotic tissue and decreased NFκB protein expression compared to control tumors, however only the HDHA+TXT had increased expression of necroptosis related proteins: RIPK1, RIPK3 and MLKL (P<.05). Increased MLKL was observed in the lipid raft portion of HDHA+TXT tumor extracts. This work confirms the efficacy of a combination therapy consisting of DHA supplementation and TXT chemotherapy using two doses of DHA as indicated by reduced tumor growth in a TNBC PDX model. Moreover, the results suggest that decreased growth may occur through increased DHA incorporation into tumor phospholipid membranes and necroptosis.