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PURPOSE Biliary tract cancers (BTCs), which include intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (EHC), and gallbladder cancer (GBC), have limited treatment options. We sought to comprehensively examine the clinical and molecular characteristics of BTCs with amplification or mutation of ERBB2. METHODS Demographic, outcome, and treatment response data were collected for patients with ERBB2-altered BTC identified by next-generation sequencing with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets from 2014 to 2018. RESULTS A total of 517 patients with BTC underwent next-generation sequencing (ICC, n = 313; EHC, n = 93; GBC, n = 111). Twenty-eight patients (5.4%) had ERBB2 alterations, including 2.7% with ERBB2 gene amplification, 2.3% with ERBB2 mutation, and 0.4% with concurrent amplification and mutation. The prevalence of ERBB2 gene alterations was significantly higher in GBC (12.6%) than in ICC (2.2%) and EHC (7.5%; P < .001). In ERBB2-amplified tumors, the median fold change was 6.4 (range, 2.1 to 19.7), while in ERBB2-mutant tumors, the most frequent mutated domain was the extracellular domain (32%), with all mutations in this region involving the S310 codon. Frequent co-altered genes in this cohort were TP53 (54%), PIK3CA (21%), and CDKN2A (18%); KRAS amplification/mutation was found in 7% of patients. One patient with ERBB2-amplified EHC who enrolled in a basket trial (ClinicalTrials.gov identifier: NCT02675829 ) had a partial response to the human epidermal growth factor receptor 2–targeted antibody-drug conjugate ado-trastuzumab emtansine. CONCLUSION ERBB2 alterations are present in 5.4% of BTCs. When present, the degree of ERBB2 gene amplification is often high, and S310 codon mutations are the most common hotspot. These features, along with the presented case, support further development of human epidermal growth factor receptor 2–targeted therapy in ERBB2-mutant and/or -amplified BTC.

Seeing double can be a good thingMany human breast cancers harbor activating mutations inPIK3CA, the gene coding for the catalytic subunit of phosphoinositide 3-kinase (PI3K). Clinical trials are underway to evaluate the efficacy of PI3K inhibitors in cancer patients. Vasanet al.found unexpectedly that a subset of breast cancers harbor not one—but two—PIK3CAmutations, and the mutations occur on the same allele (see the Perspective by Toker). In model systems, the double mutations hyperactivate PI3K signaling and enhance tumor growth. Preliminary analysis of clinical trial data suggests that breast cancers with double mutations are more responsive to PI3K inhibitors than those with a single mutation.PIK3CAmutational status could help identify the breast cancer patients most likely to benefit from these drugs.Science, this issue p.714; see also p.685

TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition1–8. With the recent approval of the first selective TRK inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors9–11. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design. A subset of patients treated with selective TRK inhibitors (including the newly approved larotrectinib) develop off-target resistance mediated by genomic acquisition of MAPK pathway-activating alterations, and may benefit from combined targeted therapy.

An understanding of the mechanisms underlying acquired resistance to cetuximab is urgently needed to improve cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Here, we present a clinical observation that MET pathway activation constitutes the mechanism of acquired resistance to cetuximab in a patient with HNSCC. Specifically, RNA sequencing and mass spectrometry analysis of cetuximab-sensitive (CetuxSen ) and cetuximab-resistant (CetuxRes ) tumors indicated MET amplification and overexpression in the CetuxRes tumor compared to the CetuxSen lesion. Stimulation of MET in HNSCC cell lines was sufficient to reactivate the MAPK pathway and to confer resistance to cetuximab in vitro and in vivo. In addition to the direct role of MET in reactivation of the MAPK pathway, MET stimulation abrogates the well-known cetuximab-induced compensatory feedback loop of HER2/HER3 expression. Mechanistically, we showed that the overexpression of HER2 and HER3 following cetuximab treatment is mediated by the ETS homologous transcription factor (EHF), and is suppressed by MET/MAPK pathway activation. Collectively, our findings indicate that evaluation of MET and HER2/HER3 in response to cetuximab in HNSCC patients can provide the rationale of successive line of treatment.

Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient-derived xenografts (PDXs) harboring amplification inHighTailored therapy with FGFRis in molecularly selected MBC based on high

Jose Manuel Baselga, renowned oncologist, died on 21 March at the age of 61 at his home in La Cerdanya, a beautiful Catalan county surrounded by the Spanish and French Pyrenees. Possibly the greatest drug developer of the past two decades, Jose was famous for, in his own words, “going to places and changing things.” Throughout his career, he transformed oncology practice and made the development of new treatments his mission. Born in Barcelona, Spain, on 3 July 1959, Jose was raised close to the Vall d'Hebron Hospital, an institution on which he would later leave his mark. He graduated from Universitat Autonoma de Barcelona in 1982 with an MD and then fulfilled his military service duty before starting his residency at the Vall d'Hebron Hospital in 1985. With a year left in his residency, Jose moved to the United States to work at Kings County Hospital in Brooklyn, New York. According to his sister Eulalia, he lived in a modest apartment with windows that would not close in winter, but the accommodations were good enough given that he was rarely home. Jose would spend his weekends and vacation time at Memorial Sloan Kettering Cancer Center (MSK) running lab experiments. He then moved full time to MSK under the mentorship of oncologists John Mendelsohn and Larry Norton. Together, they worked on newly identified antibodies targeting epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), the predecessors of the anticancer therapies cetuximab, approved for the treatment of both colorectal and head and neck cancers, and trastuzumab, which drastically changed the prognosis of HER2-positive breast cancer patients. His work at MSK also earned him a PhD from Universitat Autonoma de Barcelona in 1992. In 1996, Jose returned to Vall d'Hebron Hospital with plans to implement the MSK research model, specifically the intermingling of basic science with translationally informed oncology practice. According to some at that time, he was making a mistake moving from one of the best cancer hospitals in the United States to an unknown public hospital in Spain with no tradition in oncology research. To prove his naysayers wrong, Jose focused on what would later become a recurring pattern in his leadership: hiring the best people. Many of those he hired and trained went on to leadership roles in the oncology community. Jose knew that he also needed laboratories for basic and translational research, so he convinced Joaquin Arribas, a senior postdoc from the MSK lab of Joan Massague, to follow him and help transform an empty 70-m2 room in the middle of the oncology practice facilities into a top-notch research center. In just a few years, Jose put Barcelona and Vall d'Hebron Hospital on the cancer research map, building a bed-to-bench culture that we now call “precision medicine.” I met Jose in 2004 at Vall d'Hebron. After 40 minutes in his office, he offered me a job, but in typical fashion gave me only 72 hours to decide and begin work. I accepted in 48 hours but managed to negotiate a more leisurely start date. That decision marked the beginning of a 16-year relationship that would transform my professional and personal life. In 2010, we moved together to Boston, Massachusetts. Jose accepted the position of chief of oncology and codirector of the Massachusetts General Hospital (MGH) Cancer Center, and I followed him to build his laboratory. His vision was clear to everyone: a clinical investigation program worthy of the name of one of the world's best hospitals. Once again, Jose was not afraid of betting on young horses. Dejan Juric, a director at MGH who was then still in fellowship, became the heir-apparent to the MGH Phase I program. Astounded by Jose's drive, energy, and commitment, Dejan said to me at the time, “Mauri, I thought I was ready for anything, but I was not ready for Jose.” In January 2013, we again moved together, this time to MSK, where Jose made sure that in addition to managing his laboratory, I got my own independent group. What Jose did there in 6 years exceeded all expectations. I was in his office when, after a few days in the job, he made a phone call to someone asking the price for a biopsy (critical for translational work in clinical trials). In his way, he informed whoever was on the call that from that moment, they needed to cut the price in half. He founded and then pushed the Center for Molecular Oncology and the Early Drug Development Service to become the gems of precision medicine they are today. Jose expanded his vision by hiring physicians and scientists to build MSK-IMPACT, the targeted exome sequencing platform capable of genomically characterizing virtually every patient who walks into the clinic. Meanwhile, he empowered a young oncologist named David Hyman with the mandate and the resources to develop the best Phase I unit possible. In September 2018, Jose resigned from MSK after it was revealed that he had failed to disclose ties to drug companies. Although Jose's scientific achievements were never called into question, this incident highlighted the need for a long-overdue debate about the uniformity and clarity of conflict disclosures in medical and scientific journals. In what would become his final challenge, Jose brought his experience and stamina to AstraZeneca to reform oncology R&D in his singular manner. He immediately hired new talented clinicians and researchers to enhance what was already a successful team and built a perfectly oiled machine to test and approve new cancer drugs. Jose was direct, tough, demanding, and unforgiving, but he was also funny and caring. He would often display many or all of these characteristics in the same day. He hated mediocrity and pushed everyone around him to strive for perfection. Directly or indirectly, Jose helped the development and approval of a dozen drugs. In doing so, he saved and improved the lives of hundreds of thousands of cancer patients around the world.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Kα inhibitor alpelisib in ER+ PIK3CA mutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, e.g., MCL1 inhibitors, was experimentally validated in ER+ breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line-specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL-2 family members were highly expressed. Based on these results, newly developed cell line-specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance.

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.

The efficacy of the highly selective RET inhibitor selpercatinib is now established in RET-driven cancers, and we sought to characterize the molecular determinants of response and resistance. We find that the pre-treatment genomic landscape does not shape the variability of treatment response except for rare instances of RAS-mediated primary resistance. By contrast, acquired selpercatinib resistance is driven by MAPK pathway reactivation by one of two distinct routes. In some patients, on- and off-target pathway reactivation via secondary RET solvent front mutations or MET amplifications are evident. In other patients, rare RET-wildtype tumor cell populations driven by an alternative mitogenic driver are selected for by treatment. Multiple distinct mechanisms are often observed in the same patient, suggesting polyclonal resistance may be common. Consequently, sequential RET-directed therapy may require combination treatment with inhibitors targeting alternative MAPK effectors, emphasizing the need for prospective characterization of selpercatinib-treated tumors at the time of monotherapy progression.

Significance We report on X-ray and cryo-EM structural studies on the assembly of the human Shieldin complex composed of SHLD2, SHLD3, and REV7, as well as its complex with bound TRIP13 toward understanding the principles underlying TRIP13-mediated disassembly of Shieldin. Our studies identify a conformational heterodimeric alignment of open (O) and closed (C) conformers of REV7 when bound to a fused SHLD2–SHLD3 construct. The AAA+ ATPase TRIP13 captures the N terminus of C-REV7 (REVNT) within its central hexameric channel, with the rotatory motion associated with sequential ATP hydrolysis within individual TRIP13 subunits. This facilitates the stepwise pulling of the REV7NT through the central channel, resulting in initial disassembly of C-REV7 followed by dissociation of the Shieldin complex., The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA+ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3–REV7 binary and fused SHLD2–SHLD3–REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2–SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2–SHLD3–REV7–TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

Highlights • Canakinumab is an IL-1β antibody that neutralizes the activity of IL-1β. • The effects of canakinumab in patients with COVID-19-related pneumonia were studied. • 33 patients received canakinumab and 15 received institutional standard of care. • Treatment with canakinumab rapidly restored normal oxygen status. • Canakinumab was also associated with favorable prognosis versus SoC., Objectives Canakinumab is an IL-1β antibody that neutralizes the activity of IL-1β. We studied the efficacy and safety of canakinumab in patients with moderate COVID-19-related pneumonia. Design The aim of our study was to evaluate the reduction in duration of hospitalization with adequate oxygen status. Forty-eight patients with moderate COVID-19-related pneumonia were asked to participate in the prospective case-control study; 33 patients (Cases) signed informed consent and received canakinumab (Cohort 1); 15 patients (Controls) refused to receive the experimental drug and received institutional standard of care (SoC), (Cohort 2). Results Hospital discharge within 21 days was seen in 63% of patients in Cohort 1 vs. 0% in Cohort 2 (median 14 vs 26 days, respectively; p

On-target resistance to next-generation TRK inhibitors in TRK fusion–positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. Significance: In TRK fusion–positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation. This article is highlighted in the In This Issue feature, p. 1

Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer. Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance. RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented. Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth. Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.

Breast cancer leads current cancer diagnoses worldwide, accounting for 1 in 4 cancer cases and 1 in 6 cancer deaths in women. Over 70% of breast tumors are hormone receptor positive and can be treated with endocrine therapy, however tumor recurrence remains a major obstacle. Oncogenic mutations in the catalytic subunit of PI3K occur in up to 40% of ER+/HER2- negative breast tumors, making it a promising therapeutic target. Clinically, PI3K inhibitors have shown limited efficacy as monotherapy due to a number of resistance mechanisms, including an upregulation of ER-dependent signaling. Identification of the ER-PI3K signaling crosstalk led to clinical trials which resulted in the approval of the PI3K inhibitor alpelisib in combination with fulvestrant in patients with hormone-refractory ER+/PIK3CA-mutant breast cancer. Further work uncovered a key role for the chromatin modifier KMT2D in regulating ER-dependent transcription upon PI3K inhibition. PI3K effectors AKT1/SGK1 negatively regulate KMT2D through direct S1331 phosphorylation. When PI3K is inhibited, this downstream regulatory event is lost, leading to increased chromatin recruitment of KMT2D and an upregulation of ER-dependent transcription. We have identified a novel methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. Knockdown of SMYD2 sensitizes ER+/PIK3CA mutant breast cancer cell lines to PI3K inhibition. The combination of SMYD2 knockdown and alpelisib leads to a significant reduction in tumor growth compared to alpelisib alone in a xenograft model. Mechanistically, SMYD2 knockdown is able to attenuate alpelisib-induced KMT2D chromatin binding globally and at ER loci. Accordingly, loss of SMYD2 abrogates alpelisib-induced changes in gene expression, including ER-dependent transcription. Altogether our results demonstrate a role for epigenetic therapy using SMYD2 inhibitors in combination with alpelisib for the treatment of ER+/PIK3CA mutant breast cancer. Citation Format: Ryan Blawski, Mirna Sallaku, Xinyu Guo, Srushti Kittane, Maurizio Scaltriti, Minkui Luo, Eneda Toska. SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2955.

Pancreatic ductal adenocarcinomas (PDACs) have enhanced nutrient uptake requirements and rapid metabolic processing. The enzyme UDP-glucose pyrophosphorylase 2 (UGP2) rests at the convergence of multiple metabolic pathways, however the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an essential role for UGP2 in the maintenance of PDAC growth in both in vitro and in vivo tumor models. Transcription of UGP2 is directly regulated by the YAP/TEAD complex. Loss of UGP2 leads to decreased intracellular glycogen and defects in N-glycosylation targets important for cell growth including epidermal growth factor receptor (EGFR). In murine xenograft models, knockdown of UGP2 halted tumor growth and repressed expression of EGFR. The critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer new avenues of therapy for otherwise intractable PDACs.Impact StatementConvergent findings reveal that UDP-glucose pyrophosphorylase 2 has a central role in growth and metabolism of pancreatic ductal adenocarcinomas, highlighting novel therapeutic possibilities for this deadly cancer.

Summary PIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER+ breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[18F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance., Graphical Abstract, Highlights • Imaging hyperpolarized [1-13C]pyruvate metabolism detects response to PI3Kα inhibitors • PI3Kα inhibitors downregulate FOXM1 expression in ER+ breast tumor models • FOXM1 induces LDH expression and 13C label exchange between pyruvate and lactate • Imaging [1-13C]pyruvate metabolism could be used clinically to guide treatment, Ros et al. find that imaging of hyperpolarized [1-13C]pyruvate metabolism allows early detection of response and resistance to PI3Kα inhibition in PIK3CA-mutant ER+ breast cancer, which could be used clinically to guide treatment. Increased expression of FOXM1 results in resistance to PI3Kα inhibition and sustains LDH expression.

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) breast cancer patients. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2 and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacological inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance.

Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial’s primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. A promising approach to tackle the cancer drug resistance problem is to build mechanistic mathematical models of the signaling network of cancer cells, and explicitly model the dynamics of information flow through this network under distinct genetic conditions and in response to perturbations.In this work, we used a network-based mathematical model to identify sensitivity factors and drug combinations for the PI3Kα inhibitor alpelisib, which was recently approved for ER+PIK3CAmutant breast cancer. We experimentally validated the model-predicted efficacious combination of alpelisib and BH3 mimetics (e.g. MCL1 inhibitors) in ER+ breast cancer cell lines. We also experimentally validated the reduced sensitivity to alpelisib caused by FOXO3 knockdown, which is a novel potential resistance mechanism. Our experimental results showed cell line-specific sensitivity to the combination of alpelisib and BH3 mimetics, which was driven by the choice of BH3 mimetics. We find that cell lines were sensitive to the addition of either MCL1 inhibitor s63845 alone or in combination with BCL-XL/BCL-2 inhibitor navitoclax, and that the need for the combination of both BH3 mimetics was predicted by the expression of BCL-XL. Based on these results, we developed cell line-specific network models that are able to recapitulate the observed differential response to alpelisib and BH3 mimetics, and also incorporate the most recent knowledge on resistance and response to PI3Kα inhibitors.Overall, we present an approach for the development, experimental testing, and refining of mathematical models, which we apply to the context of PI3Kα inhibitor drug resistance in breast cancer. Our approach predicted and validated PI3Kα inhibitor sensitivity factors (FOXO3 knockdown) and drug combinations (BH3 mimetics), and illustrates that network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance.

Somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (Patients were enrolled on the basis of localA total of 166 patients withTaselisib had limited activity in the tumor types tested and is no longer in development. This genome-driven study improves understanding of the activity, limitations, and resistance mechanisms of using PI3K inhibitors as monotherapy to target

Treatment paradigms for patients with upper tract urothelial carcinoma (UTUC) are typically extrapolated from studies of bladder cancer despite their distinct clinical and molecular characteristics. The advancement of UTUC research is hampered by the lack of disease-specific models. Here, we report the establishment of patient derived xenograft (PDX) and cell line models that reflect the genomic and biological heterogeneity of the human disease. Models demonstrate high genomic concordance with the corresponding patient tumors, with invasive tumors more likely to successfully engraft. Treatment of PDX models with chemotherapy recapitulates responses observed in patients. Analysis of a HER2 S310F-mutant PDX suggests that an antibody drug conjugate targeting HER2 would have superior efficacy versus selective HER2 kinase inhibitors. In sum, the biological and phenotypic concordance between patient and PDXs suggest that these models could facilitate studies of intrinsic and acquired resistance and the development of personalized medicine strategies for UTUC patients., The advancement of upper tract urothelial carcinoma (UTUC) research is hampered by the lack of disease-specific models. Here, the authors report patient derived xenograft and cell line models of UTUC, and show that these models retain the genomic and biological heterogeneity of human disease.

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

Background Kinase fusions are rare and poorly characterized in breast cancer (BC). We aimed to characterize kinase fusions within a large cohort of advanced BC. Patients and methods A total of 4854 patients with BC were analyzed by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) targeted DNAseq and MSK-Fusion targeted RNAseq during the study time period. Results Twenty-seven of 4854 (0.6%) patients harbored fusions: 11 FGFR (five FGFR2, three FGFR3, three FGFR1), five BRAF, four NTRK1, two RET, two ROS1, one ALK, one ERBB2, and one MET. A history of endocrine therapy was present in 15 (56%) of fusion-positive BC; eight of the 15 cases had available pre-treatment samples, of which six were fusion-negative. None of the fusion-positive BC samples harbored ESR1 hotspot mutations. Two patients with acquired LMNA-NTRK1 fusions and metastatic disease received larotrectinib and demonstrated clinical benefit. Conclusion Kinase fusions in BC are extremely rare, and appear to be enriched in hormone-resistant, metastatic carcinomas and mutually exclusive with ESR1 mutations. The present study expands the spectrum of genetic alterations activating mitogen-activated protein kinase (MAPK) signaling that can substitute for ESR1 mutations in this setting. Molecular testing at progression after endocrine therapy should include fusion testing, particularly in the absence of ESR1 hotspot alterations, in an effort to identify additional therapeutic options which may provide substantial clinical benefit.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Molecular profiling of personal cancer genomes, and the identification of actionable vulnerabilities and drug-response biomarkers, are the basis of precision oncology. Tumors often present several driver alterations that might be connected by cross-talk and feedback mechanisms, making it difficult to mark single oncogenic variations as reliable predictors of therapeutic outcome. In the current work, we uncover and exploit driver alteration co-occurrence patterns from a recently published in vivo screening in patient-derived xenografts (PDXs), including 187 tumors and 53 drugs. For each treatment, we compare the mutational profiles of sensitive and resistant PDXs to statistically define Driver Co-Occurrence (DCO) networks, which capture both genomic structure and putative oncogenic synergy. We then use the DCO networks to train classifiers that can prioritize, among the available options, the best possible treatment for each tumor based on its oncogenomic profile. In a cross-validation setting, our drug-response models are able to correctly predict 66% of sensitive and 77% of resistant drug-tumor pairs, based on tumor growth variation. Perhaps more interesting, our models are applicable to several tumor types and drug classes for which no biomarker has yet been described. Additionally, we experimentally validated the performance of our models on 15 new tumor samples engrafted in mice, achieving an overall accuracy of 75%. Finally, we adapted our strategy to derive drug-response models from continuous clinical outcome measures, such as progression free survival, which better represent the data acquired during routine clinical practice and in clinical trials. We believe that the computational framework presented here could be incorporated into the design of adaptive clinical trials, revealing unexpected connections between oncogenic alterations and increasing the clinical impact of genomic profiling.

BackgroundAlthough the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance.MethodsMouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment.ResultsUsing preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+T cells. Activation of CD8+T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME.ConclusionOur findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor–host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.

Breast cancer (BC) is the second most common cause of cancer-related deaths in women worldwide. The availability of reliable biomarkers of response/resistance to cancer treatments would benefit patients and clinicians allowing for a better selection of BC patients most likely to respond to a specific treatment. Phosphatidylinositol 3-kinase (PI3K) enzymes are involved in numerous cellular- functions and processes. The gene encoding for PI3K catalytic subunit p110α is mutated in 20-40% of BC. We performed a meta-analysis of the current literature on randomized clinical trials, investigating the role of PIK3CA mutational status as prognostic factor and predictor of response to anti-cancer treatments. Overall 1929 cases were included. The pooled analysis confirmed that the presence of a PIK3CA mutation represents an independent negative prognostic factor (HR = 1.67, 95% CI: 1.15-2.43; p = 0.007) in BC, as previously reported. Since PI3K signalling is also a result of other pathways’ hyperactivation, further investigation of potential biomarkers able to predict likelihood of response to anti-PI3K/mTOR, anti-HER2 and other TKRs is warranted in future randomized clinical trials. This article is protected by copyright. All rights reserved

Background: Epidermal growth factor receptor (EGFR) is expressed in ˜50% of triple negative breast cancer (TNBC) and has been proposed as a therapeutic target in this disease. However, trials testing EGFR blockade in TNBC failed to show significant clinical benefit. Probable reasons for such results were patient selection based on EGFR expression and the enrollment of heavily pretreated metastatic patients. Our team has conducted two neoadjuvant trials testing the activity of the anti-EGFR antibodies panitumumab (PTB) and cetuximab (CTX) combined with chemotherapy in locally advanced TNBC. Biomarkers predictive of pathological complete response (pCR) were the level of tumor cell EGFR protein expression and tumor-infiltrating lymphocytes' (TILs) profile (PMIDs 24827135, 26649807). The PTB-treated pts had a higher pCR rate (47%) than the CTX-treated pts (24%), but also a twice higher relapse rate, after 5 years of follow-up. Here we report results of genomic and TILs studies, performed in order to reveal possible determinants of recurrences in those trials. Methods: Tumor tissues sampled before and after neoadjuvant therapy (NAT) have been analyzed by next generation sequencing (NGS) using a targeted exome panel (MSK-IMPACT) of 410 cancer-related genes. Gene expression was evaluated by Affymetrix arrays. TIL density was assessed on pre-NAT samples according to Salgado et al, 2014 (PMID 25214542). The correlation between response, recurrences, genomic and TIL findings was analyzed in a case-by-case fashion. Results: Sixteen tumors that achieved pCR (PTB: 11, CTX: 5) and 23 non-pCR tumors (PTB: 11, CTX: 12) have been analyzed. For 14 non-pCR tumors (PTB: 6, CTX: 8) data have been obtained both from the pre-NAT and the post-NAT sample. Among those tumors, 6 recurred within 2 years after surgery (PTMB: 3, CTX: 3) and assays are on-going on several others that relapsed. Several genomic aberrations that potentially played a causative role in opposing to therapy were identified. We observed multiple mutations in the PI3K pathway in several non-pCR or relapsing pts. Interestingly, in a residual tumor (RT) of a non-pCR patient we found 3 different activating mutations in PIK3CA and one in PTEN. Another example of genomic selection induced by pharmacological pressure is the emergence of a HRAS G12S mutation in a RT after CTX. Additional novel findings include in-frame mutations and deletions in ARID1B and PARP1 amplification in non-pCR pts. Most of the tumors which recurred had ≤10% TILs (9/13) and only 4/13 had ≥30%. Among the tumors with a post-NAT RT but without recurrence, 17/33 had ≤10% TILs and 16/33 ≥30%. No particular link between TIL density and mutation pattern was observed. Conclusions: This is an example of a case-by-case approach where we captured the intrinsic inter-tumor heterogeneity, which is likely responsible for the different responses to EGFR-targeting in TNBC. Genes/pathways candidate of resistance to therapy are currently being validated. Citation Format: Radosevic-Robin N, Cocco E, Privat M, Abrial C, Penault-Llorca F, Scaltriti M. Potential recurrence markers of locally advanced triple negative breast cancer treated by combined neoadjuvant EGFR targeting and chemotherapy, revealed by genomic analyses and assessment of tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-29.

The incidence of esophagogastric cancer is rapidly rising, but only a minority of patients derive durable benefit from current therapies. Chemotherapy as well as anti-HER2 and PD-1 antibodies are standard treatments. To identify predictive biomarkers of drug sensitivity and mechanisms of resistance, we implemented prospective tumor sequencing of patients with metastatic esophagogastric cancer. There was no association between homologous recombination deficiency defects and response to platinum-based chemotherapy. Patients with microsatellite instability–high tumors were intrinsically resistant to chemotherapy but more likely to achieve durable responses to immunotherapy. The single Epstein–Barr virus–positive patient achieved a durable, complete response to immunotherapy. The level of ERBB2 amplification as determined by sequencing was predictive of trastuzumab benefit. Selection for a tumor subclone lacking ERBB2 amplification, deletion of ERBB2 exon 16, and comutations in the receptor tyrosine kinase, RAS, and PI3K pathways were associated with intrinsic and/or acquired trastuzumab resistance. Prospective genomic profiling can identify patients most likely to derive durable benefit to immunotherapy and trastuzumab and guide strategies to overcome drug resistance. Significance: Clinical application of multiplex sequencing can identify biomarkers of treatment response to contemporary systemic therapies in metastatic esophagogastric cancer. This large prospective analysis sheds light on the biological complexity and the dynamic nature of therapeutic resistance in metastatic esophagogastric cancers. Cancer Discov; 8(1); 49–58. ©2017 AACR. See related commentary by Sundar and Tan, p. 14. See related article by Pectasides et al., p. 37. This article is highlighted in the In This Issue feature, p. 1

Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.

Background: Bruton's tyrosine kinase (BTK) inhibition is a key component in B-cell receptor signaling and is one of the most prominent therapeutic targets in hematologic malignancies. Acalabrutinib is a highly selective, covalent, potent next-generation inhibitor of BTK (Barf et al, 2017) and is approved for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia (CLL). However, a small subset of patients develop resistance to acalabrutinib over time. While the major mechanism of resistance to covalent BTK inhibitors is explained by acquired mutations in BTK-Cys481 and its downstream target PLCg2, additional mechanisms are under investigation. Previously, we presented that high surface expression of CD49d (VLA-4) and CD79B correlates with acalabrutinib resistance in CLL patients (Bibikova et al, 2019). In the present study, we performed optimized whole genome methylation sequencing on 42 clinical samples from 22 patients in the ACE-CL-001 clinical trial (NCT02029443) to better understand novel mechanisms of acalabrutinib resistance at an epigenetic level in CLL patients. Methods: DNA was extracted from peripheral blood mononuclear cells of patients with available samples at baseline (n=20), following acalabrutinib treatment at the time of progressive disease (n=8), or at 6 months post treatment start (n=14, used as non-progressor controls). While bisulfite conversion has been the gold standard for DNA methylation analysis for decades, it damages DNA and performs suboptimally with clinical FFPE and cfDNA samples that are often in limited quantity. Enzymatic methods have recently been reported that do not induce DNA damage, making them more suitable to routine clinical samples that are already DNA-damaged (eg, by formalin fixation) and often have limited DNA amounts. Before performing methylome analysis, we conducted a comprehensive comparison of bisulfite and enzymatic DNA methylation assays in reference cell lines and clinically relevant samples . This revealed that enzymatic methods outperformed bisulfite in several library quality metrics and resulted in increased library yield with the same DNA input and PCR amplification cycles. Enzymatic methods also captured a larger fraction of GC-rich regions, including CpG islands, in all comparisons. Prior to interrogating the full CLL 22 patient sample set, the enzymatic methods were validated in a subset of 6 CLL samples. We conducted a comprehensive analysis of methylation data sets with previously established RNA-seq analysis from paired samples. Results: Analysis of the acalabrutinib posttreatment samples revealed that the non-progressed patients had significantly higher methylation of 440 genes, whereas progressed samples had higher methylation of only 2 genes. CDKN1C, IL15, and AXL were significantly more methylated in non-progressed samples (Figure 1A), and these genes have been shown to be related to proliferation, survival, or response to BTK inhibition in CLL (De Totero et al, 2008; Sinha et al, 2018; Gupta et al, 2019; Lu et al, 2021; Kagiyama et al, 2021). Using RNA-seq data from paired samples, we showed that, as expected, IL15 methylation negatively correlated with IL15 expression (Figure 1B; R=−0.73, p=7.1e-09). IL15 is a proinflammatory cytokine that has downstream signaling targets in the NF-kB pathway, similar to BTK, indicating that alternative methods of NF-kB signaling may affect response. To support this hypothesis, we found that IL15 methylation significantly correlated with BIRC2 (R=0.59, p=1.4e-5) and RELA (R=0.42, p=0.0034) expression. Both genes play a role in NF-kB signaling, where BIRC2 encodes for the protein cIAP1 that regulates NF-kB activation and RELA encodes for p65, a member of the NF-kB transcription factor family. To our knowledge, it is not known if DNA methylation of IL15-induced expression plays a role in CLL resistance to BTK inhibition, and this finding may have important implications for future combination treatments. To note, CDKN1C, IL15, and AXL methylation were also significantly different between the progressed and non-progressed patients before acalabrutinib treatment, indicating they may be playing a role in innate resistance to acalabrutinib treatment. Conclusions: Using EM-seq to analyze DNA methylation, we found that reduced IL15 methylation and increased IL15 expression correlates with acalabrutinib innate resistance in CLL patients. Figure 1 Figure 1. Disclosures Burke: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Nuttall: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company. Karl: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Callahan: AstraZeneca: Current Employment; Veritas Genetics: Current holder of individual stocks in a privately-held company. Mendler: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months; BISC Global, Inc.: Ended employment in the past 24 months. Criscione: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Naumenko: AstraZeneca: Consultancy. Bibikova: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months; Acerta Pharma: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Bloecher: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Dougherty: AstraZeneca: Current Employment, Current equity holder in publicly-traded company, Research Funding; Pfizer: Current equity holder in publicly-traded company. Barrett: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Research Funding. Scaltriti: AstraZeneca: Current Employment, Current holder of stock options in a privately-held company; MSKCC: Research Funding. Cingolani: AstraZeneca: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Furman: Oncotracker: Consultancy; Sunesis: Consultancy; Janssen: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy; Beigene: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Incyte: Consultancy; Abbvie: Consultancy, Honoraria, Other: Expert testimony; AstraZeneca: Honoraria; Morphosys: Consultancy; Sanofi: Consultancy; X4 Pharmaceuticals: Consultancy. Brown: Invectys: Other: Data Safety Monitoring Committee Service; TG Therapeutics: Research Funding; Beigene: Consultancy; Genentech/Roche: Consultancy; MEI Pharma: Consultancy; Morphosys AG: Consultancy; Bristol-Myers Squib/Juno/Celegene: Consultancy; Rigel: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Catapult: Consultancy; Acerta/Astra-Zeneca: Consultancy; Eli Lilly and Company: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Nextcea: Consultancy; Loxo/Lilly: Research Funding; Sun: Research Funding; SecuraBio: Research Funding; Gilead: Research Funding. Mortlock: ARTICA Therapeutics B.V.: Membership on an entity's Board of Directors or advisory committees; Anavo Therapeutics: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Hadfield: AstraZeneca: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months. Munugalavadla: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

(ER)-positive tumors, also known as luminal breast cancers represent over 70% of breast cancers and they are treated primarily with endocrine therapy. Despite the success of endocrine therapies, resistance to these agents is a major clinical problem, and it eventually develops in all patients with metastatic disease. Therefore, the identification and understanding of the mechanisms of resistance to endocrine therapy is a major research priority. Important insights into the mechanisms of endocrine resistance have been gained with the identification of recurrent, activating mutations in ESR1 (the gene coding ER) in approximately 18 % of the tumors with acquired resistance to aromatase inhibitors. However, the mechanisms of resistance in the remaining 82 % of the cases largely unknown. We have recently genomically characterized the largest cohort of endocrine resistant metastatic breast tumors (n=1918 tumors). In addition to ESR1 mutations, we identified the presence of inactivating mutations in the ARID1A gene of the SWI/SNF complex in metastatic endocrine-resistant breast tumors. In parallel, as part of our efforts to elucidate mechanisms of resistance to endocrine therapy, we performed an epigenome CRISPR/CAS9 knockout screen that identified ARID1A as the top candidate whose loss determined resistance to the ER degrader in the clinic fulvestrant. We validated these findings showing that ARID1A knockout cells are resistant to endocrine therapy in vitro and in vivo. Hence, from both clinical observations and our unbiased CRISPR screen from our laboratory, we identified ARID1A loss as a candidate of endocrine resistance. We next performed for the first time the mechanistic characterization of ARID1A loss in ER-positive breast cancer. We determined that ARID1A loss remodels the chromatin landscape of breast cancer on a genome-wide scale, converging to a basal-like gene expression program resulting from downregulation of master transcription factors (TFs) that are required for luminal (ER+) identity. Transcriptome profiling of ARID1A knockout cell lines and patient samples with loss of ARID1A were consistent with our initial findings showing an induction of basal-like gene expression programs. Thus, we elucidated a new mechanism of endocrine resistance through lineage switching mediated by the inactivation of ARID1A. Mechanistically, we showed that ARID1A-dependent SWI/SNF complex binding was impaired at the genomic sites of the major luminal-lineage determining TFs including ER, FOXA1, and GATA3. We also determined that ARID1A regulates ER-chromatin interactions and ER-dependent transcription, the defining driver in ER+ breast cancer. Hence, tumors cells with loss of ARID1A shift from ER-dependent luminal cells to ER-independent basal-like cells and become less responsive to luminal specific anti-ER therapy. Altogether, we uncovered a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER+ breast cancer. This work which I am the last and corresponding author of was recently published to Nature Genetics (Xu et al, Nature Genetics 2020).Interestingly, further analyses of Arid1a knockout in the mammary gland and breast organoids validated a key role of ARID1A in mammary gland morphogenesis and luminal cell differentiation. Moreover, single cell RNA-seq analyses demonstrated that ARID1A knockout breast cells did not differentiate to mature luminal cells. In the cohort of breast cancer tumors (n=1918), we also identified the presence of hotspot missense mutations in the pioneer transcription factor FOXA1 to be enriched in metastatic endocrine-resistant breast tumors. Mutations in the pioneer transcription factor FOXA1 are a hallmark of ER-positive lobular breast cancers. However, the functional consequences of FOXA1 mutations and whether they affect the therapeutic response to endocrine therapy are currently unknown. We undertook a comprehensive approach to investigate the structural consequences, including the genome-wide chromatin recruitment, chromatin accessibility, and transcriptional network in breast cancer models harboring recurrent hotspot and breast cancer specific FOXA1 mutations. By examining the landscape of FOXA1 mutations in a larger cohort (4,952 breast cancer patients), we identified several hotspot mutations in the Wing2 region, and a breast-cancer specific mutation located in the third beta strand, namely SY242CS. Interestingly, FOXA1 hotspot missense mutations were found to be more common in metastatic samples compared to primary tumors, and mutually exclusive with ESR1 mutations, suggesting a positive correlation between presence of FOXA1 mutations and resistance to endocrine therapy. Indeed, FOXA1 mutations were associated with a lower response to aromatase inhibitors in breast patient samples. Mechanistically, we uncovered that FOXA1 Wing2 mutations display increased chromatin binding affinity at ER sites upon estrogen stimulation, and enhanced ER-mediated transcription. Accordingly, 3D structural molecular simulations determined a highly organized 3D conformation conferred by FOXA1 mutations in the Wing2 loop, which may drive augmented ER recruitment or potentially allow more stable/durable interactions with ER and other cooperating factors at the chromatin. By contrast, FOXA1 SY242CS displayed a novel pioneering function over distinct genomic regions that were enriched by the presence of a new FOXA1-binding motif. This alternative binding motif was FOXA1 SY242CS-specific and common at both unique accessible and chromatin bound sites, leading to novel open chromatin regions and the induction of an alternative transcriptome regardless of estrogen stimulation, granting this variant a cellular growth advantage. Thus mechanistically, two phenotypic groups of FOXA1 mutants were established: hypermorphic Wing2 mutants that augment estrogen response, and a neomorphic SY242CS mutant that promotes an alternative pioneering, and cistromic and transcriptomic function leading to endocrine therapy resistance. This work which I am a corresponding author of was recently published to Cancer Cell Arruabarrena-Aristorena et al, Cancer Cell 2020). In summary, our data provide insights into how mutations in transcriptional regulators perturb their function to dictate breast cancer progression and therapeutic response. Our work highlights the power of examining recurrent cancer-associated mutations to advance our understanding of transcriptional regulators in breast cancer. These results also provide mechanistic insights into how FOXA1 and ARID1A mutations are associated with worse outcome to endocrine therapy and position these genetic alterations as potential biomarkers of endocrine therapy response for the treatment of metastatic ER+ breast cancer. Citation Format: Eneda Toska, Guotai Xu, Amaia Arruabarrena-Aristorena, Maurizio Scaltriti. Epigenetic mechanisms of endocrine therapy response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG05.

Background Acquisition of resistance to anticancer therapies is associated with increased expression of immunosuppressor modulators that enable tumor cells to escape from the antitumor immunity machinery. Objective To understand the mechanisms of immune escape from MEK inhibitors in head and neck cancer (HNC). Methods We have developed several mitogen-activated protein kinase (MAPK) pathway-driven cancers in immunocompetent mice. Specifically, a 4NQO (tobacco surrogate) carcinogenesis induced HNC model, novel KRASG12-driven HNC model using CRISPR technology, and HRASV12 model using a lentiviral vector. These models were used to study mechanisms of immune escape from trametinib, an MAPK pathway inhibitor. Results All of these HNC models are sensitive to trametinib in vitro and in vivo; however, over time, resistance was developed, and tumor progression was detected. In this work, we found that chronic treatment with trametinib induced epithelial-mesenchymal transition and upregulation of the tumor-derived immunosuppressor modulators like PD-L1. The tumor ecosystem was also altered during the acquisition of resistance because upon short treatment with trametininb during stable disease, tumors were enriched with infiltrated CD8+ T, whereas upon progression the CD8+ T cells in the tumors showed an exhausted phenotype with high levels of programmed cell death protein 1 (PD-1). Blocking the immune escape mechanisms using anti PD-1/ programmed death ligand 1 (PD-L1) drugs together with trametinib induced tumor eradication and most mice were cured. Conclusions Our findings suggest that administration of blockers of the PD1/PD-L1 axis enhance the antitumor activity of MAPK-targeted drugs and delay the appearance of resistance.

One of the hallmarks of hormone receptor (HR)-positive breast cancer is its dependence on the phosphatidylinositol-3-kinase (PI3K) pathway. Here, we review the epidemiologic, functional, and pharmacologic interactions between oncogenic PI3K and the estrogen receptor (ER). We discuss the epidemiology of PI3K pathway alterations, mechanisms of resistance to PI3K inhibitors, and the current mechanistic landscape of crosstalk between PI3K and ER, which provide the rationale for dual ER and PI3K inhibition and is now a standard of care in the treatment of ER+ PIK3CA-mutant metastatic breast cancer. We outline newer studies in this field that delineate the clinically relevant overlaps between PI3K and parallel signaling pathways, insulin signaling, and ER epigenetic modifiers. We also identify several caveats with the current data and propose new strategies to overcome these bottlenecks.

PURPOSE: The activating mutation AKT1(E17K) occurs in ~7% of ER+ metastatic breast cancer (MBC). We report, from a multipart, first-in-human, Phase I study (NCT01226316), tolerability and activity of capivasertib, an oral AKT inhibitor, as monotherapy or combined with fulvestrant in expansion cohorts of AKT1(E17K)-mutant ER+ MBC patients. PATIENTS AND METHODS: Patients with an AKT1(E17K) mutation, detected by local (NGS) or central (plasma-based BEAMing) testing, received capivasertib 480 mg bid, 4 days on, 3 days off, weekly or 400 mg bid combined with fulvestrant at the labeled dose. Study endpoints included safety, objective response rate (ORR; RECIST v1.1), progression-free survival (PFS) and clinical benefit rate at 24 weeks (CBR(24)). Biomarker analyses were conducted in the combination cohort. RESULTS: From October 2013 to August 2018, 63 heavily pretreated patients received capivasertib (20 monotherapy, 43 combination). ORR was 20% with monotherapy, and within the combination cohort was 36% in fulvestrant-pretreated and 20% in fulvestrant-naïve patients, although this latter group may have had more aggressive disease at baseline. AKT1(E17K) mutations were detectable in plasma by BEAMing (95%, 41/43), ddPCR (80%, 33/41) and NGS (76%, 31/41). A ≥50% decrease in AKT1(E17K) at cycle 2 day 1 was associated with improved PFS. Combination therapy appeared more tolerable than monotherapy (most frequent grade ≥3 adverse events: rash [9% vs 20%], hyperglycemia [5% vs 30%], diarrhea [5% vs 10%]). CONCLUSIONS: Capivasertib demonstrated clinically meaningful activity in heavily pretreated AKT1(E17K)-mutant ER+ MBC patients, including those with prior disease progression on fulvestrant. Tolerability and activity appeared improved by the combination.

Purpose: AZD5363/capivasertib is a pan-AKT catalytic inhibitor with promising activity in combination with paclitaxel in triple-negative metastatic breast cancer harboring PI3K/AKT-pathway alterations and in estrogen receptor–positive breast cancer in combination with fulvestrant. Here, we aimed to identify response biomarkers and uncover mechanisms of resistance to AZD5363 and its combination with paclitaxel. Experimental Design: Genetic and proteomic markers were analyzed in 28 HER2-negative patient-derived xenografts (PDXs) and in patient samples, and correlated to AZD5363 sensitivity as single agent and in combination with paclitaxel. Results: Four PDX were derived from patients receiving AZD5363 in the clinic which exhibited concordant treatment response. Mutations in PIK3CA/AKT1 and absence of mTOR complex 1 (mTORC1)-activating alterations, for example, in MTOR or TSC1, were associated with sensitivity to AZD5363 monotherapy. Interestingly, excluding PTEN from the composite biomarker increased its accuracy from 64% to 89%. Moreover, resistant PDXs exhibited low baseline pAKT S473 and residual pS6 S235 upon treatment, suggesting that parallel pathways bypass AKT/S6K1 signaling in these models. We identified two mechanisms of acquired resistance to AZD5363: cyclin D1 overexpression and loss of AKT1 p.E17K. Conclusions: This study provides insight into putative predictive biomarkers of response and acquired resistance to AZD5363 in HER2-negative metastatic breast cancer.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Pre-existent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively these data define HER2 mutations as a therapeutic target in breast cancer and suggest that co-existence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib.

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1–10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.

Summary: The PI3K pathway integrates extracellular stimuli to phosphorylate effectors such as AKT and serum-and-glucocorticoid-regulated kinase (SGK1). We have previously reported that the PI3K pathway regulates estrogen receptor (ER)-dependent transcription in breast cancer through the phosphorylation of the lysine methyltransferase KMT2D by AKT. Here, we show that PI3Kα inhibition, via a negative-feedback loop, activates SGK1 to promote chromatin-based regulation of ER-dependent transcription. PI3K/AKT inhibitors activate ER, which promotes SGK1 transcription through direct binding to its promoter. Elevated SGK1, in turn, phosphorylates KMT2D, suppressing its function, leading to a loss of methylation of lysine 4 on histone H3 (H3K4) and a repressive chromatin state at ER loci to attenuate ER activity. Thus, SGK1 regulates the chromatin landscape and ER-dependent transcription via the direct phosphorylation of KMT2D. These findings reveal an ER-SGK1-KMT2D signaling circuit aimed to attenuate ER response through a role for SGK1 to program chromatin and ER transcriptional output. : Toska, Castel, et al. show that the PI3K pathway propagates its effects to control chromatin and estrogen receptor (ER) function through SGK1, a PI3K effector. PI3K inhibitors, via a negative-feedback loop, activate SGK1, which phosphorylates the histone lysine methyltransferase KMT2D to attenuate its activity and regulate ER response. Keywords: SGK1, KMT2D, PI3K pathway, estrogen receptor, breast cancer, chromatin regulation, AKT, PI3K inhibitors

Activating mutations in

On-target resistance to next-generation kinase inhibitors, drugs specifically designed to maintain potency in the setting of kinase domain mutations, remains poorly characterized. Having identified that xDFG (TRKA G667) mutations confer second-generation TRK inhibitor resistance in patients with TRK fusion-positive cancers, we used computational modeling and biochemical assays to study how these affect drug binding. We found that xDFG mutations stabilize the inactive kinase conformation, hampering the binding of type I second-generation TRK inhibitors, while increasing the affinity for type II multikinase inhibitors. Type II drugs consistently inhibit the growth and TRK-mediated signaling of isogenic and patient-derived models harboring xDFG mutations. Collectively these data demonstrate that TRK xDFG substitutions trigger conformational changes that favor type II binding mechanisms. Given prior identification of paralogous xDFG resistance mutations in other oncogene addicted cancers, these findings provide insight into rational drug design to address these recalcitrant resistant alterations by leveraging inhibitor class affinity switching.

Background NTRK1, NTRK2 and NTRK3 fusions are present in a plethora of malignancies across different histologies. These fusions represent the most frequent mechanism of oncogenic activation of these receptor tyrosine kinases, and biomarkers for the use of TRK small molecule inhibitors. Given the varying frequency of NTRK1/2/3 fusions, crucial to the administration of NTRK inhibitors is the development of optimal approaches for the detection of human cancers harbouring activating NTRK1/2/3 fusion genes. Materials and methods Experts from several Institutions were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) to review the available methods for the detection of NTRK gene fusions, their potential applications, and strategies for the implementation of a rational approach for the detection of NTRK1/2/3 fusion genes in human malignancies. A consensus on the most reasonable strategy to adopt when screening for NTRK fusions in oncologic patients was sought, and further reviewed and approved by the ESMO TR and PM WG and the ESMO leadership. Results The main techniques employed for NTRK fusion gene detection include immunohistochemistry, fluorescence in situ hybridization (FISH), RT-PCR, and both RNA-based and DNA-based next generation sequencing (NGS). Each technique has advantages and limitations, and the choice of assays for screening and final diagnosis should also take into account the resources and clinical context. Conclusion In tumours where NTRK fusions are highly recurrent, FISH, RT-PCR or RNA-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK1/2/3 fusions are uncommon, either front-line sequencing (preferentially RNA-sequencing) or screening by immunohistochemistry followed by sequencing of positive cases should be pursued.

Dysregulation of the PI3K/AKT/mTOR pathway has the potential to trigger the activation of class I phosphoinositide 3-kinases (PI3Ks) and eventually lead to cancer. Class I PI3Ks are heterodimers composed of the regulatory subunit p85 and the catalytic subunit p110. There are four isoforms of p110, alpha and beta, important in solid tumors, and delta and gamma, preferentially expressed in leukocytes, and drivers in hematological malignancies. While activation of PI3K p110alpha is the most frequent oncogenic event in solid tumors, p110beta plays an important role in PTEN deficient cancers and in mediating resistance to p110alpha selective PI3K inhibitors.MEN1611 is a PI3K inhibitor active on p110alpha (both mutants and wt), beta and gamma (8.6- and 2.2- fold less potent compared to the alpha, respectively), while sparing the delta isoform (36-fold less potent compared to the alpha). Thus, MEN1611 differs from other selective PI3K inhibitors such as alpelisib (p110alpha selective) and taselisib (p110beta sparing). MEN1611 is currently in clinical development for patients with HER2 positive advanced or metastatic breast cancer in combination with trastuzumab with/without fulvestrant (B-PRECISE-01). Patient-derived xenograft (PDX) models and breast cancer cell lines with different genetic background were used to assess the antitumor activity of MEN1611 in p110 alpha- and beta- dependent tumors. Healthy-donors derived CD69+ B-cells, which are primarily dependent on p110 delta for proliferation and survival were used to test the activity of the compound against the p110delta. According to the biochemical selectivity, MEN1611 demonstrated a lower cytotoxic potential in a p110delta-driven cellular model (CD19+ B-cells), when compared to taselisib (>150-fold) and an improved cytotoxic activity in the p110beta-driven cellular model (PTEN-null breast cancer cells) when compared to alpelisib. Moreover, MEN1611 selectively decreases the p110alpha protein levels in PIK3CA mutated breast cancer cells in a concentration-dependent manner, with a corresponding reduction in pAKT. In vivo, MEN1611 monotherapy showed significant and long-lasting anti-tumor activity, reflected in tumor-stasis or tumor regression in several trastuzumab-resistant PIK3CA-mutant HER2 positive PDXs with different concomitant genomic aberrations. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. Interestingly, MEN1611 monotherapy showed a significant tumor volume inhibition in a PTEN-null breast cancer PDX model. Overall, our data provide evidence that MEN1611 is active against HER2 positive and PTEN-null breast tumors. Moreover, its delta-sparing profile may have potential implications on MEN1611 tolerability and ability to overcome resistance to p110alpha selective inhibitors. Citation Format: Alessio Fiascarelli, Corrado Carrisi, Giuseppe Merlino, Stefania Capano, Diego Bisignano, Simone Talucci, Alessandro Bressan, Daniele Bellarosa, Mario Bigioni, Maurizio Scaltriti, Joaquin Arribas, Andrea Pellacani, Massimiliano Salerno, Monica Binaschi. Men1611 is a pi3k α/β selective and δ sparing inhibitor with long-lasting antitumor activity in different genetic backgrounds of pik3ca mutant breast cancer models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-13.