AIMS:Resistance to anti-HER2 therapies, particularly trastuzumab, remains a major obstacle in the treatment of HER2-positive (HER2 +) breast cancer. This study aims to uncover novel mechanisms driving trastuzumab resistance with a focus on the immune component, key mediator of trastuzumab efficacy. METHODS:We developed an isogenic cell line-derived xenograft model to perform transcriptome-wide analyses of trastuzumab-sensitive and -resistant tumors. To validate key findings, we employed a 3D cancer-immune co-culture system capable of quantifying antibody-dependent cellular cytotoxicity (ADCC). RESULTS:Transcriptomic profiling revealed how trastuzumab treatment shifts tumor transcriptomes, including changes that remodel the metabolic landscape and distinct gene signatures associated with resistance, notably the upregulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Functional studies demonstrated that PFKFB3 promotes trastuzumab resistance by inducing metabolic rewiring and reducing ADCC. Silencing PFKFB3 restored immune-mediated cytotoxicity. Clinical dataset analyses confirmed that elevated PFKFB3 expression correlates with reduced overall and progression-free survival, and with incomplete pathological response to trastuzumab. CONCLUSIONS:PFKFB3 upregulation drives metabolic adaptations that confer resistance to trastuzumab in HER2 + breast cancer. These findings highlight PFKFB3 as a promising therapeutic target to overcome resistance and improve patient outcomes.

Background: Patient-derived organoids (PDOs) generated from benign breast tissue and breast carcinomas have successfully recapitulated their in vivo counterparts. PDOs model tumorigenesis and allow for screening novel therapeutics personalized to individual patients. However, acquiring cells to generate PDOs is cumbersome. This study demonstrates the feasibility of fine-needle aspiration biopsy (FNAB) for harvesting cells for PDOs modeling ductal carcinoma in situ (DCIS) and compares the efficacy with core needle biopsy (CNB). Methods: Surgical specimens from patients with biopsy-proven DCIS were used for this study. CNB was performed on fresh specimens in the operating room, and tissue was mechanically dissociated before culture in basement membrane extract (BME) and organoid medium to generate PDOs. FNAB was performed in the pathology gross room on fresh specimens, and the aspirate was similarly submitted for culture. Results: PDOs were successfully generated in 15 of 18 specimens obtained by CNB and seven of 11 specimens obtained by FNAB. The average time to initial organoid growth was 4 days for FNAB specimens compared to 19.3 days for CNB specimens. Tumor cells were seen on seven of 11 FNAB smears and 16 of 18 CNB touch preps. Immunofluorescence staining confirmed the presence of both luminal and myoepithelial cells in derived PDOs. Conclusions: FNAB effectively obtains cells for PDOs modeling DCIS. CNB yielded PDOs with a high success rate, but they were slow to establish. The time to organoid growth was significantly shorter for FNAB specimens. Thus, FNAB offers an efficient alternative for breast PDO culture and can reduce the time and resources spent on generating PDO cultures.

Background Patient-derived organoids (PDOs) generated from benign breast tissue and breast carcinomas have successfully recapitulated their respective in vivo counterparts. PDOs model tumorigenesis and allow for screening of novel therapeutics personalized to individual patients. However, acquiring cells to generate PDOs is cumbersome. We demonstrate the feasibility of fine needle aspiration biopsy (FNAB) for harvesting cells for PDOs modeling ductal carcinoma in situ (DCIS). Methods Surgical specimens from patients with biopsy-proven DCIS were used for this study. Core needle biopsy (CNB) was performed on fresh specimens in the operating room, and tissue was mechanically dissociated before culture in basement membrane extract (BME) and organoid medium to generate PDOs. FNAB was performed in the gross room on fresh specimens, and the remaining aspirate was similarly submitted for PDO culture. Results PDOs were successfully generated in 15/18 specimens obtained by CNB and 7/11 specimens obtained by FNAB. The average time to initial organoid growth was 4 days for FNAB specimens compared to 19.3 days for CNB specimens. Tumor cells were seen on 7/11 FNAB smears and 16/18 CNB touch preps. Immunofluorescence staining confirmed the presence of both luminal and myoepithelial cells in derived PDOs. Conclusions FNAB effectively obtains cells for PDOs modeling DCIS. CNB after mincing yielded PDOs with a high success rate, but they were slow to establish. Notably, the time to organoid growth was significantly shorter for FNAB specimens. Thus, FNAB offers an efficient alternative for breast PDO culture and can reduce the time and resources spent on generating PDO cultures. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R01CA281361 UCSF Resource Allocation Program

Abstract Cancer progression remains a major obstacle to the successful treatment of cancer. In HER2+ breast cancer, targeting therapies against HER2 have revolutionized the treatment landscape. However, predictive factors for response are largely unknown and a significant number of patients develop resistance. An extensive body of work has revealed a plethora of mechanisms of resistance. However, a key aspect of cancer cells has been largely overlooked: their aberrant glycosylation profile. Aberrant glycosylation of proteins is a hallmark in cancer and has been linked to multiple processes such as invasion, angiogenesis and modulation of the immune response. Yet the influence of altered glycosylation on the efficacy of HER2-targeted therapies remains unknown. Therefore, here, we address this question using clinical data, 3D co-culture systems and advanced live microscopy. Bioinformatics analysis of SPY-2 clinical trial data reveals an association of specific glycogenes with response to HER2-targeting therapies in breast cancer patients. We validated these findings in resistance cell models generated in-vitro and in-vivo using gain and loss of function assays. Furthermore, to provide a more relevant cell model system, we implement the use of 3D co-cultures of cancer spheroids and immune cells. Thus, our research addresses a largely unexplored area in cancer biology combining clinical data from tumor biopsies, trastuzumab-resistant cancer cell models and, live imaging of 3D heterotypic cultures. This work holds the potential to identify new markers of response to therapies and highlights the importance of the interplay of cancer cells and their microenvironment on the efficacy of current treatments. Citation Format: Ana Ruiz-Saenz, Roos Vincken, Denise Wolf, Veronica Steri, Laura van't Veer, John Martens, Danny Huylebroeck, Mark M. Moasser. Tumor glycosylation impacts the efficacy of HER2 targeting therapies [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 2014.

This file contains supplementary figures including the western blots corresponding to the schematics of figures 2,3,4,5, as well as additional data sets referred to in the paper.

We hypothesized that eribulin combined with cyclophosphamide (EC) would be an effective combination with tolerable toxicity for the treatment of advanced breast cancer (ABC). Patients with histologically confirmed metastatic or unresectable ABC with any number of prior lines of therapy were eligible to enroll. In the dose escalation cohort, dose level 0 was defined as eribulin 1.1 mg/m2 and cyclophosphamide 600 mg/m2, and dose level 1 was defined as eribulin 1.4 mg/m2 and cyclophosphamide 600 mg/m2. Eribulin was given on days 1 and 8 and cyclophosphamide on day 1 of a 21-day cycle. In the dose expansion cohort, enrollment was expanded at dose level 1. The primary objective was clinical benefit rate (CBR), and secondary objectives were response rate (RR), duration of response (DOR), progression-free survival (PFS), and safety. No dose-limiting toxicities were identified in the dose escalation cohort (n = 6). In the dose expansion cohort, an additional 38 patients were enrolled for a total of 44 patients, including 31 patients (70.4 ≥ 5 CTC/7.5 mL) at baseline. Median PFS was shorter in patients who were CTC-positive vs. negative (13.1 vs 30.6 weeks, p = 0.011). In heavily pretreated patients with ABC, treatment with EC resulted in an encouraging CBR of 79.5

Figure S1 - A) HCC1569-HER3WT and HCC1569-HER3KO immunoblots, soft agar assays, and FISH assays. Figure S2 - Phospho-RTK array legend and darker exposures Figure S3 - H&E and Ki67 stains of HCC1569KO tumors Figure S4 - Confirmation of human specificity of anti-vimentin Ab Figure S5 - Lack of expression of HER family members in NR6 cells Figure S6 - p85 isoform expression in cell lines and Ras knockdowns in HCC1569 cells Figure S7 - Prolonged HER3 knockdown in HCC1569 cells Figure S8 - expression of HER2 YF mutants

Supplementary Data from Phosphorylation of the src Epithelial Substrate Trask Is Tightly Regulated in Normal Epithelia but Widespread in Many Human Epithelial Cancers

PDF file - 77K, Src activity in SmoM2 induced skin neoplasia

The oncogene ERBB2 encoding the receptor tyrosine-protein kinase erbB-2 (HER2) is frequently overexpressed or amplified and occasionally mutated in a variety of human cancers. The early discovery of this oncogene, its established oncogenic relevance in diverse cancers, its substantial expression on the surface of cancer cells, and its druggable catalytic activity have made it one of the most pursued targets in the history of cancer drug development. Initiatives targeting HER2 provided the early stimulus for several transformational pharmaceutical technologies, including mAbs, tyrosine kinase inhibitors, antibody-drug conjugates, and others. The seismic impact of these efforts has been felt in treatment of many cancers, including breast, gastroesophageal, lung, colorectal, and others. This impact continues to broaden with increasing indications on the horizon and a plethora of novel agents in development. However, implementation of these therapeutic strategies has been complex. The clinical translation of every one of these classes of agents has been notable for underperformance or overperformance characteristics that have informed new lines of research providing deeper insights into the mechanistic complexities and unrealized opportunities provided by this molecular target. Despite all the successes to date, the preponderance of scientific evidence indicates that the full potential of HER2 as a target for cancer therapeutics is far greater than currently realized, and numerous lines of investigation are ongoing to deepen and broaden the scope of impact of HER2 as a signaling, homing, or immunologic target. In this review, we explore the existing data and evolving paradigms surrounding this remarkable target for cancer therapy.

PDF file - 1099K, p-Trask immunostain of wounded mouse skin

ABSTRACTBRAFV600Emutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF/MEK/EGFR co-targeting, we used a high throughput kinase activity mapping platform. We found that SRC kinases are systematically activated in BRAFV600ECRC following targeted inhibition of BRAF ± EGFR, and that coordinated targeting of SRC with BRAF ± EGFR increases efficacyin vitroandin vivo. SRC drives resistance to BRAF ± anti-EGFR therapy independently of ERK signaling by inducing transcriptional reprogramming via beta-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2-loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF+EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft (PDX) models. COX2 inhibition represents a novel drug-repurposing strategy to overcome therapeutic resistance in BRAFV600ECRC.

1091 Background: Talimogene laherparepvec (TVEC) is a modified oncolytic herpes simplex 1 (HSV1) virus that may enhance tumor immune infiltration and is currently FDA-approved for the treatment of unresectable cutaneous, subcutaneous, and nodal melanoma. Anti-tumor responses have been seen both locally and systemically, and an abscopal systemic effect has been described in distant organ metastases. Methods: In this single arm, open label Phase 1b study, patients received intra-tumoral TVEC (first dose 10 6 PFU/mL followed by 10 8 PFU/mL q2-3 weeks; volume based on tumor size up to max 4mL) in combination with CT (gemcitabine/carboplatin [GC], nab-paclitaxel [Nab-P], or paclitaxel [P]) or ET at the discretion of the treating physician. All patients had at least one 1 cm lesion that was injectable at the bedside. The primary endpoint was safety and tolerability. The secondary endpoint was response by RECIST 1.1. Blood and tissue-based immune correlates including injected and neighboring non-injected lesions were evaluated. Results: 19 pts were enrolled on this study (2/5/20 – 1/25/23) and evaluable for toxicity with 1 pt non-evaluable for efficacy due to early discontinuation. Median age was 52.1 years. Nine pts (47%) had HR+/HER2- BC and 10 pts (53%) had TNBC. Injected lesions included intact subcutaneous skin nodules (7), non-fungating breast or chest wall lesions (12), and fungating breast or chest wall lesions (9). Pts had a median of 3 prior lines of systemic therapy in the metastatic setting (range 0-9). 13 pts (74%) had visceral metastases. Pts received TVEC with the following treatment partners: GC (n=8, 42%), Nab-P (n=7, 37%), P (n=2, 11%), ET (n=2, 10%). Median treatment duration was 11.6 weeks (range 1.0-45.0 weeks). Grade 3-4 treatment-related adverse events included neutropenia (n=5, 26%), anemia (n=1, 5%), thrombocytopenia (n=1, 5%), and injection site skin ulceration (n=1, 5%). Three pts (16%) had Grade 1-2 injection site skin ulceration. Response per RECIST 1.1 was evaluated in 16 pts (2 pts had rapid progression prior to first response evaluation): PR (n=2, 13%), SD (n=5, 31%), and PD (n=9, 56%). Disease response at the site of TVEC injection was clinically evaluated in 18 pts, with response rates (RR) as follows: partial response (PR) (n=11, 61%), stable disease (SD) (n=4, 22%), and progressive disease (PD) (n=3, 17%). Mass cytometry (CyTOF) analysis demonstrated a decrease in HLA-DR expression circulating lymphocytes and in TIM3 expression across multiple cell types in responders vs. non-responders; updated correlative analyses will be presented. Conclusions: The addition of intra-tumoral TVEC to CT or ET is safe and tolerable in pts with advanced BC. This treatment induces changes in circulating immune responses. Clinical trial information: NCT03554044 .

Supplementary table 1 Observed and predicted blood toxicity profile at the start of Cycle 2 and Cycle 4 (A) and PK-toxicity model-predicted (B) neutrophil and platelet toxicity at cycle 2, day 1 and cycle 5 talozaparib 1mg/carboplatin AUC 1/5 weekly dosing: Absolute change from baseline. Supplementary Table 2: PK toxicity model estimates Supplementary Figure 1a: Goodness of fit plots of all modeled PK and PK-toxicity data. Supplementary Figure 2 Talazoparib drug clearance in gBRCA carriers versus non-carriers of talazoparib Supplementary table 3 Simulated blood toxicity profile of new dosing regimens using the PK-toxicity model. A) planned schedule B) optimized talazoparib, C) optimized carboplatin, D) optimized combination E) simulation of single agent toxicity.

A) Rationale for study drug dose interruption or reduction; B) Rationale for cisplatin dose interruption or reduction; C) Cisplatin dose intensity, stratified by dose level

The pharmaceutical inactivation of driver oncogenes has revolutionized the treatment of cancer, replacing cytotoxic chemotherapeutic approaches with kinase inhibitor therapies for many types of cancers. This approach has not yet been realized for the treatment of HER2-amplified cancers. The monotherapy activities associated with HER2-targeting antibodies and kinase inhibitors are modest, and their clinical use has been in combination with and not in replacement of cytotoxic chemotherapies. This stands in sharp contrast to achievements in the treatment of many other oncogene-driven cancers. The mechanism-based treatment hypothesis regarding the inactivation of HER2 justifies expectations far beyond what is currently realized. Overcoming this barrier requires mechanistic insights that can fuel new directions for pursuit, but scientific investigation of this treatment hypothesis, particularly with regards to trastuzumab, has been complicated by conflicting and confusing data sets, ironclad dogma, and mechanistic conclusions that have repeatedly failed to translate clinically. We are now approaching a point of convergence regarding the challenges and resiliency in this tumor driver, and I will provide here a review and opinion to inform where we currently stand with this treatment hypothesis and where the future potential lies.

Surface-targeting biotherapeutic agents have been successful in treating HER2-amplified cancers through immuno-stimulation or chemodelivery but have failed to produce effective inhibitors of constitutive HER2-HER3 signaling. We report an extensive structure-function analysis of this tumor driver, revealing complete uncoupling of intracellular signaling and tumorigenic function from regulation or constraints from their extracellular domains (ECDs). The canonical HER3 ECD conformational changes and exposure of the dimerization interface are nonessential, and the entire ECDs of HER2 and HER3 are redundant for tumorigenic signaling. Restricting the proximation of partner ECDs with bulk and steric clash through extremely disruptive receptor engineering leaves tumorigenic signaling unperturbed. This is likely due to considerable conformational flexibilities across the span of these receptor molecules and substantial undulations in the plane of the plasma membrane, none of which had been foreseen as impediments to targeting strategies. The massive overexpression of HER2 functionally and physically uncouples intracellular signaling from extracellular constraints.