Patient-derived Xenograft Studies Research Articles

The roles of patient-derived xenograft models and artificial intelligence toward precision medicine.

Patient-derived xenografts (PDX) involve transplanting patient cells or tissues into immunodeficient mice, offering superior disease models compared with cell line xenografts and genetically engineered mice. In contrast to traditional cell-line xenografts and genetically engineered mice, PDX models harbor the molecular and biologic features from the original patient tumor and are generationally stable. This high fidelity makes PDX models particularly suitable for preclinical and coclinical drug testing, therefore better predicting therapeutic efficacy. Although PDX models are becoming more useful, the several factors influencing their reliability and predictive power are not well understood. Several existing studies have looked into the possibility that PDX models could be important in enhancing our knowledge with regard to tumor genetics, biomarker discovery, and personalized medicine; however, a number of problems still need to be addressed, such as the high cost and time-consuming processes involved, together with the variability in tumor take rates. This review addresses these gaps by detailing the methodologies to generate PDX models, their application in cancer research, and their advantages over other models. Further, it elaborates on how artificial intelligence and machine learning were incorporated into PDX studies to fast-track therapeutic evaluation. This review is an overview of the progress that has been done so far in using PDX models for cancer research and shows their potential to be further improved in improving our understanding of oncogenesis.

Abstract CT083: A first-in-human phase 1/2a study of a novel anti-PAUF monoclonal antibody PBP1510 in patients with advanced/metastatic pancreatic cancer

Abstract Background Pancreatic Adenocarcinoma Up-regulated Factor (PAUF) is a protein overexpressed in pancreatic cancer but not in non-cancerous tissues and plays an important role in carcinogenesis and metastasis. PBP1510 is a first-in-class, anti-PAUF humanized immunoglobulin G1 monoclonal antibody developed for treatment of pancreatic cancer. PBP1510 demonstrated promising pancreatic cancer growth inhibition efficacies in in vivo patient-derived xenograft studies and is being evaluated in humans for the first time in the ongoing PAUF-I study. Methods PAUF-I (NCT05141149) is an open-label, multi-center, Phase 1/2a study in advanced/metastatic pancreatic cancer patients. Eligible patients must have a tumor that has progressed on standard chemotherapy, with at least 1 measurable lesion per RECIST v1.1 criteria, an ECOG performance status score of 0 or 1, a life expectancy of ≥ 3 months, and adequate baseline organ function to enroll. PBP1510 is administered as a 90-minute intravenous infusion in 28-day cycles and tumor assessments are performed every 8 weeks from the first dose. Phase 1 uses a 3 + 3 dose-escalation design to assess the safety, tolerability, pharmacokinetics (PK), and immunogenicity of ascending doses of PBP1510 as a single agent or in combination with gemcitabine (Table 1). The observation period for dose-limiting toxicities (DLT) is 28 days. ORR and pre- and post-treatment PAUF levels in tumor tissue will be explored in the analyses. A recommended phase 2 dose (RP2D) will be selected based on PK, safety, and efficacy data from Phase 1. Phase 2a is the dose expansion phase which aims to evaluate the safety, tolerability, PK, immunogenicity, and efficacy of PBP1510 administered at RP2D in combination with gemcitabine. As of January 2024, 6 patients have been enrolled. The 1M cohort (1 mg/kg) was completed without any report of DLT. Dose escalation is in progress and DLT evaluation is ongoing for the 2nd and 3rd patients in the 2M cohort. TABLE 1: NAND Dose escalation and cohorts (Phase 1) Monotherapy Combination Cohort PBP1510 Dose (mg/kg) Gemcitabine Dose (mg/m2) Cohort PBP1510 Dose (mg/kg) Gemcitabine Dose (mg/m2) 1M 1 - 1C 1 1000 2M 3 - 2C 3 1000 3M 6 - 3C 6 1000 4M 10 - 4C 10 1000 5M 15 - 5C 15 1000 Citation Format: Jaime Feliu Batlle, Daniel A. King, Jamie Kim, Sumita Pradhan, Yun-Yong Park, Kedar Diwakar Mandakhalikar, Wei Qi Loh. A first-in-human phase 1/2a study of a novel anti-PAUF monoclonal antibody PBP1510 in patients with advanced/metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT083.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

BackgroundOvarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and...

Abstract 7151: Targeting metabolic pathway in triple negative breast cancer with a small molecule inhibitor

Abstract Background: Triple Negative Breast Cancer (TNBC) is the most aggressive type of breast cancer, making up 15-20% of diagnosed cases, and has high rates of metastasis and recurrence. Most women with this disease experience a recurrence after curative-intent therapy for early breast cancer, while a smaller proportions are diagnosed with distant metastases initially. Cancer cells are characterized by their reprogramming of glucose metabolism. Cancer cells show increased proliferation along with higher energy and substrate demands. Through our efforts to find effective therapies for TNBC, we have identified CET019, a new small molecule that effectively suppresses the growth of breast cancer cells. Methods: Cell viability and clonogenic assays were performed on a range of TNBC cells, including MDA-MB-231, MDA-MB-468, SUM159, HCC1395, HCC1806, EMT6, and 4T1. Cell cycle distribution was assessed through flow cytometry. LC-MS/MS analysis was performed on TMT-labeled samples for global proteome profiling. The efficacy of CET019 (30 mg/kg) was tested using tumor and patient derived xenograft studies. Results: Our studies show that CET019 suppresses the clonogenic potential and growth of various triple-negative breast cancer cells. The CET019 treatment causes cell cycle arrest in TNBC cells at the G2/M phase. Proteomics analysis suggests that CET019 specifically affects metabolic and proteosomal pathways. In vivo studies with CET019 found an inhibition of tumor growth kinetics in the mouse xenograft study with different TNBC models, as compared to the control group. We also observed that CET019 inhibited metastatic lung nodules. The results were further substantiated in three Patient Derived Xenograft (PDX) models. Conclusion: In summary, our study has identified a novel small molecule candidate CET019 as a promising lead candidate for TNBC treatment, paving the way for further development.“The complete chemical structures of the compounds used will be disclosed at the time of presentation at the meeting.” Citation Format: Arpit Dheeraj, Fernando Jose Marques, Dhanir Tailor, Abel Bermudez, Benedikt Grau, Sharon Pitteri, Sanjay V. Malhotra. Targeting metabolic pathway in triple negative breast cancer with a small molecule inhibitor [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 7151.

Abstract 4250: Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research

Abstract Introduction: The development of new drugs in cancer therapy comprises toxicity and efficacy tests with increasing complexity. First and foremost, in vitro experiments are performed with well-established cancer cell lines, which are subsequently validated in animal experiments as prerequisite for clinical trials. Until a few years ago, there were gaps in the complexity chain between in vitro and in vivo experiments. Ethical considerations have led to use of systems like organ on a chip or mini-organ 3D in vitro models. In cancer research, cell cultures or organoids are generated from patient tumor material. However, these cultures only reflect the tumor of one patient, which is why panels of patient tumors should be used to evaluate the effectiveness of drugs on different patients. In vivo panel screens of patient derived xenografts (PDX) mouse models need high numbers of animals and takes several months. A pre-screen with in vitro models generated from in vivo PDX tissues allows large scale and faster pre-screens complementing in vivo systems for more focused in vivo analyses. Methods: Currently we have a pool of more than 600 established PDX models of 21 tumor entities. From this pool, cancer tissues of glioblastoma, mesothelioma, gastric, head/neck, lung and breast cancer were processed in single cell suspensions and cultured under defined conditions to obtain adherent cells or spheroids. The generated PDX in vitro cultures were analyzed for cellular impurities, cancer stem cell content and perpetuation of in vivo PDX characteristics. FACS analyses for tumor specific markers, chemo sensitivity assays and growth characteristics of the PDX derived cell lines (especially for glioblastoma) were analyzed. Results: From PDX tissues used, 90% grew as adherent and/or spheroid PDX derived in vitro cultures, in which mouse cells were entirely depleted. A high percentage of these cultures showed enriched cancer stem cell features and stem cell marker expression. Tumor marker expression and standard drug sensitivity data correlate to the in vivo PDX and derived in vitro cell culture models. RNAseq data were used to predict drug sensitivities in silico for untested drugs and drug combinations on our newly established PDX derived glioblastoma cell lines. Initial screens with predicted candidates were performed. Promising conditions were successfully repeated in corresponding animal PDX models. Conclusion: The newly developed technology for establishment if in vitro cell cultures from PDX efficiently generates stably growing cell lines possessing all key features of the original PDX. These cell lines can be used for initial pre-screens to optimize and improve selection of pharmacologically active drugs or drug combinations before initiating in vivo PDX studies. Citation Format: Lars Winkler, Joshua Alcaniz, Maria Stecklum, Wolfgang Walther, Jens Hoffmann. Adherent and spheroid cell models of patient-derived xenograft for drug development and translational research [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 4250.

Abstract 6904: Model-derived progression-free survival (mPFS): A better metric for patient-derived xenograft studies

Abstract Patient-derived xenografts (PDX) are a powerful assay system for translational prediction of therapeutic potential for experimental therapeutics. The primary readout from PDX models is tumor shrinkage, measured by the ratio of treated/control tumor volume. This readout corresponds loosely to the Objective Response Rate (ORR) metric used in clinical trials, in that it is a readout of drug effect on the sensitive subclones within a tumor. In prior work, we have shown that there is a paradoxical relationship between short-term tumor response and progression-free survival (PFS). While ORR is the primary measure utilized in the Phase II to III transition in clinical development, approval hinges on survival metrics, and the disconnect between ORR and PFS creates a potential risk of late-stage failures for experimental anticancer therapeutics. Thus, it is of interest to understand whether there are alternative approaches to analyzing PDX data that can enable the assessment of therapeutic potential in terms of a projected PFS. In this simulation study, we will examine the accuracy of progression-free survival (PFS) estimates obtained directly from observation during a PDX study and compare their accuracy with the accuracy of PFS estimates derived using a mathematical model-based approach that relies on a kinetic model of tumor subclone growth (model-based PFS, or mPFS) fitted directly to PDX tumor growth and rebound kinetics under treatment. We generated our test dataset using a mathematical model of tumor kinetic growth to simulate xenograft data using fitted parameters derived from literature, adding in experimental error consistent with prior experimental measurement-derived variability. We used the simulated raw data to calculate an observed PFS as well as mPFS derived from fitting the tumor clonal kinetic model to the raw data in a blinded fashion. Our results show that the empirical PFS from PDX models is subject to overestimation, as a function of the observation time interval. On the other hand, mPFS is significantly less biased and more accurate, but is subject to some logistical constraints on sampling. Our work demonstrates the practical translational utility of a novel analysis method (mPFS) in deriving estimates of progression-free survival from PDX model data. Citation Format: Andrew Chen, Madison Stoddard, Lin Yuan, Debra Van Egeren, Arijit Chakravarty, Dean Bottino. Model-derived progression-free survival (mPFS): A better metric for patient-derived xenograft studies [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 6904.

Therapeutic benefit of the dual ALK/FAK inhibitor ESK440 in ALK-driven neuroblastoma

Neuroblastoma (NB) is a predominantly pediatric cancer with greater than 90% of cases arising in children under the age of five. More than half of patients have metastases detected at diagnosis, and high-risk disease is associated with five-year survival rates of only 50–60 %. Standard therapy involves highly toxic chemotherapy, surgery, radiation, and immunotherapy, and less toxic, more specific targeted therapies are urgently needed. Genomic studies have identified common driver aberrations in high-risk NB, such as MYCN amplification. In addition, a proportion of high-risk patients harbor amplification or activating mutations in anaplastic lymphoma kinase (ALK), and co-occurrence of ALK mutations and MYCN amplification have been associated with aggressive disease. In this study, we analyzed the efficacy of a Phase Ia-cleared, orally bioavailable dual ALK and focal adhesion kinase (FAK) inhibitor, ESK440, in multiple preclinical NB models. ESK440 potently inhibited proliferation of NB cell lines, with increased sensitivity in cell lines harboring ALK aberrations. ALK, FAK, and downstream target activation were rapidly decreased upon ESK440 treatment, and this was associated with impaired cellular migration and invasion. Importantly, ESK440 treatment also decreased MYCN levels. NB cell line and patient-derived xenograft studies showed significant reduction in tumor growth in ESK440-treated mice with no signs of toxicity. In certain NB models, ESK440 showed comparable or enhanced efficacy to lorlatinib, another clinical ALK inhibitor, and a lorlatinib-resistant cell line (COG-N-561 LR) retained sensitivity to ESK440. These preclinical results indicate that ESK440 is a promising targeted agent for ALK-driven NB and support future clinical studies to evaluate its efficacy in NB patients.

Abstract C115: Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate

Abstract STRO-002 is a novel folate receptor alpha (FolRα)-targeting antibody-drug conjugate (ADC) currently undergoing evaluation in clinical trials at various phases of development in ovarian and endometrial cancer and pediatric AML. Here, we describe the favorable properties of the STRO-002 ADC and summarize its activity in preclinical models. STRO-002 is an ADC composed of an anti-FolRα antibody conjugated to four tubulin-targeting hemiasterlin warheads. Incorporation of non-natural amino acids, using Sutro’s XpressCF® and XpressCF+® cell-free expression and conjugation systems, allows for site-specific conjugation of the hemiasterlin payload, yielding a homogenous and high-fidelity ADC. As an ADC, STRO-002 internalizes rapidly into FolRα+ tumor cells and exhibits potent target-dependent cell killing on xenograft tumor cell lines. Further characterization of STRO-002 and the hemiasterlin payload reveals significant ADC bystander activity and a lower sensitivity to drug efflux via the P-gp drug pump compared to other microtubule-targeting warheads. In addition to robust direct and bystander cell killing, STRO-002 is also capable of triggering immunogenic cell death (ICD). In in vitro cell killing assays, STRO-002 induces all three hallmarks of ICD—HMGB1 and ATP release and surface exposure of calreticulin—and in in vivo vaccination studies, injection of STRO-002-killed tumor cells was effective at establishing a protective immune response against subsequent tumor challenge, identifying STRO-002 as a bonafide ICD inducer.In vivo, STRO-002 treatment demonstrates robust efficacy in ovarian xenograft models, even when treatment is initiated in large, established tumors. In addition, combination therapy with STRO-002 and carboplatin, anti-VEGF, or anti-PD-L1 results in greater efficacy than treatment with single agents alone, demonstrating combination benefit with current standard-of-care therapies. To better understand the activity of STRO-002 in more-translatable preclinical models, we turned to patient-derived xenografts (PDX) and found that STRO-002 exhibits good anti-tumor activity in endometrial cancer and non-small cell lung cancer PDX studies, suggesting potential clinical benefit in these indications. In conclusion, STRO-002 exhibits multiple advantageous properties as an ADC and shows good anti-tumor activity, either in combination or as a single agent, in both cell-derived xenograft and PDX models. Citation Format: Robert Yuan, Andrew McGeehan, Sihong Zhou, Jennifer Smith, Dayson Moreira, Krishna Bajjuri, Cuong Tran, Gang Yin, Alice Yam, Helena Kiefel, Xiaofan Li. Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C115.

Abstract A002: The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies

Abstract Large PDX collections provide broad representation of tumor genetics and are commonly used to evaluate the clinical potential of novel therapeutic strategies. In many solid malignancies, profound benefit in PDX preclinical studies has foreshadowed eventual FDA approval of clinically impactful therapeutics. With a lack of corresponding progress in glioblastoma (GBM), the value of PDX testing has been questioned. Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients, and over the past 23 years, we have used this collection for extensive in vivo preclinical testing of 145 drugs in both orthotopic and heterotopic settings. As part of this work, the impact of standard of care therapy with temozolomide (TMZ) and bevacizumab was evaluated in orthotopic models using clinically relevant dosing regimens. For each animal study, the ratio of median survival for treated vs. control animals were used to define a ‘survival ratio’ as a normalized metric of benefit. TMZ efficacy was evaluated in 26 newly diagnosed GBM PDXs, with equal representation of MGMT methylated and unmethylated tumors. As expected, MGMT methylated PDXs were much more sensitive to TMZ as compared to unmethylated PDXs (median survival ratio 4.47 vs 1.48, respectively), and similar to clinical experience, 2 MGMT unmethylated PDXs were markedly sensitive to TMZ, while 1 MGMT methylated PDX was relatively resistant. In a more limited study, dose-dense vs. standard TMZ dosing was compared in 7 PDXs (4 MGMT methylated, 3 MGMT unmethylated). In a pooled analysis of all lines, both standard (HR=0.09; 95% CI: 0.06–0.14) and dose-dense TMZ therapy (HR=0.09; 95% CI: 0.06–0.14) were superior to vehicle controls (p<0.001). Similar to the clinical results in the Phase III randomized RTOG 0525 trial, survival with the dose-dense TMZ was no different than standard dosing (p=0.88, HR=0.98; 95% CI: 0.72-1.32). In an analysis of bevacizumab across 32 PDXs, mice were dosed once a week until moribund, and a minimum overall median survival benefit of 20% was used to define ‘responders’. Thirteen PDXs met this criterion, although the overall survival ratio among this subset was limited (range 1.21-1.65). Only 2 models had a 50% or greater survival benefit. Stratification by disease state (24 newly diagnosed vs 8 recurrent) showed equivalent performance (median survival benefit 1.13 for both). These results are consistent with the limited survival benefit observed with bevacizumab and the RTOG 0825 clinical trial. In summary, the results from these relatively inexpensive PDX studies demonstrate the potential of pre-clinical PDX trials to evaluate the extent of benefit and fraction of responsive PDXs to a novel therapeutic. If analyzed across an ‘adequate’ number of PDXs, these results potentially can be used to identify predictive biomarkers or to compare various treatment regimens prior to clinical testing. Citation Format: Danielle M Burgenske, Ann C Mladek, Katrina K Bakken, Zeng Hu, Brett L Carlson, Paul A Decker, Jeanette E Eckel-Passow, Jann N Sarkaria. The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A002.

Targeting CREB-Binding Protein (CREBBP) Overcomes Resistance to Azacitidine and Venetoclax Therapy in Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is a clonal myeloid malignancy arising from hematopoietic stem or progenitor cells (HSPC) within the bone marrow (BM). Despite advancements in our understanding of AML pathogenesis, patients continue to face grim outcomes, with a five-year relative survival rate of approximately 20%. This highlights the pressing need to identify molecular features of AML that are amenable to therapeutic intervention. The ideal molecular targets would be those broadly dysregulated and integral to multiple signaling pathways implicated in AML pathogenesis. Recent therapeutic regimens have incorporated venetoclax, a small molecule inhibitor of BCL2, in combination with hypomethylating agents, showing moderate success. Venetoclax (VEN) acts as a BH3 mimetic, specifically inhibiting BCL2. While clinical outcomes of VEN combined with hypomethylating agents (decitabine or azacitidine - AZA) have been promising, a substantial subset of patients remains refractory or experiences relapse following treatment with these regimens. Due to the molecular and cellular heterogeneity of AML, identifying ideal mechanisms to circumvent resistance remains elusive. In our investigations, we have observed that patient-derived cells from AML exhibit a higher frequency of dysregulated immune and inflammatory pathways than previously appreciated, referred to as “oncogenic immune signaling states.” Furthermore, immune and inflammatory-related pathways are preferentially expressed in AML patients who are resistant/refractory to VEN/AZA compared to those who respond favorably. To identify critical immune and inflammatory-related pathways contributing to VEN/AZA responses, we developed an oncogenic immune signaling-targeted CRISPR-library, aimed at identifying potential therapeutic targets involved in VEN/AZA resistance in AML. Utilizing this targeted CRISPR-screen approach, we successfully identified CREB-binding protein (CREBBP) as the top candidate linked to resistance to VEN/AZA therapy. Notably, CREBBP alterations have been associated with a poor prognosis in de novo AML, making it a compelling target for further investigation. To validate CREBBP's role in VEN/AZA resistance, we treated a VEN/AZA-resistant AML cell line, THP1 (FAB subtype M5, KMT2A-r), with commercially available pharmacologic inhibitors of CREBBP, A485, and CCS1477, and assessed the response. Remarkably, inhibition of CREBBP with either A485 or CCS1477 significantly decreased cell proliferative ability and clonogenic potential. Importantly, co-treatment with either A485 or CCS1477 and VEN/AZA synergistically abrogated cell proliferation and clonogenic potential of THP1 cells, effectively overcoming resistance (Figure 1). Our ongoing studies include validating CREBBP as a potential therapeutic target for overcoming VEN/AZA resistance in AML. We have created CREBBP-knockdown cell lines through genetic manipulation (shRNA) and will utilize these to confirm the specificity of CREBBP in modifying resistance to VEN/AZA. In addition we will utilize patient-derived xenograft studies in immunocompromised (NRG) mouse models. By investigating CREBBP's role in mediating resistance, we aim to unlock new avenues for enhancing the efficacy of VEN/AZA therapy in AML patients, particularly those facing resistance to current therapies. In conclusion, our research highlights the critical involvement of CREBBP in VEN/AZA resistance in AML. Targeting this molecular pathway holds the promise of offering a potential therapeutic approach to improve treatment outcomes for AML patients, particularly those facing resistance to current therapies. Understanding and harnessing CREBBP's influence could pave the way for developing personalized and effective strategies to combat this challenging hematological malignancy.

Calibrated Release IL15 Bivalent CD33 and/or FLT3 and NOT Emcn Logic Gated Gene Circuit CAR-NK Cell Therapy (SENTI-202) in Venetoclax Resistant Patient Derived Xenograft Acute Myeloid Leukemia Models

Venetoclax (VEN) as combination therapy has improved response rates and overall survival of patients with acute myeloid leukemia (AML). However, once AML relapse prognosis is dire with a 5.3 month median survival (Brandwein, 2020). While chimeric antigen receptor (CAR) cell therapies have revolutionized the treatment landscape for lymphoid hematological malignancies, development of such therapies for AML has been challenging, in part due to the heterogeneity of the disease. Currently, there is a paucity of individual AML targets that are consistently expressed across AML subpopulations. Furthermore, the expression of these AML targets is not restricted to tumor cell populations, often resulting in off-tumor toxicity against healthy cell populations. SENTI-202 employs OR and NOT Logic Gating to overcome these challenges. SENTI-202 represents an innovative preclinical CAR-NK cell therapy, engineered to exploit a powerful CD33 OR FLT3 NOT EMCN Logic Gate gene circuit, in conjunction with calibrated release IL-15 (crIL15) expression. The CD33 OR FLT3 (OR GATE) activating CAR (aCAR) concurrently targets two AML antigens, thus permitting a broader therapeutic window against AML LSCs (CD33+/-, FLT3+), blasts (CD33+, FLT3+/-), and more specifically, VEN-resistant (VEN-r) AML with a CD33+ monocytic phenotype (Pei, 2020). The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) is a pivotal safeguard, selectively shielding healthy EMCN+ Hematopoietic Stem Cells and Progenitor Cells (HSCs/PCs) from potential off-tumor toxicity. Previously, SENTI-202 exhibited remarkable efficacy in targeting and eliminating CD33 and/or FLT3 expressing AML cell lines, while concurrently safeguarding healthy HSCs/PCs (Garrison et al., ASH, 2022). To further validate the efficacy and broaden the utility of SENTI-202, we conducted assessments on clinically relevant preclinical AML models, along with VEN-r AML patient-derived xenograft (PDX) models. We first assessed the in vitro cytotoxicity and cytokine profiles of CAR-NK cells possessing the SENTI-202 OR Gate and crIL15 against primary R/R AML patient samples (n=11), where we observed significantly higher cytotoxicity compared to unengineered NK-cells (68% vs 19.7%) (p=0.0005, E:T ratio 1:1 for 24h) and increased TNF-a and IFN-g secretion. To further explore CAR-NK cell anti-tumor activity, we screened 10 different PDX samples and discovered that while the median frequency of CD33 and/or FLT3 expression was 99.9% (97.48-100), individual CD33 and FLT3 expression level varied, underscoring AML proteomic heterogeneity and the clinical need for OR Gate approaches. Our initial PDX studies used clinically relevant VEN/HMA-r AML PDX cells (designated as PDX1) obtained from a patient with FLT3-ITD, GATA2, and NRAS mutations, who initially responded to VEN/decitabine treatment but later relapsed. To facilitate in vivo tracking of AML and disease progression, the VEN-r PDX cells were engineered with Akaluc, an exceptionally brighter luciferase compared to conventional luciferase. Following confirmation of AML engraftment in bone marrow through bioluminescence imaging (BLI), the CAR-NK cells, along with unengineered NK-cells, were infused at a dose of 30 million cells per infusion, once a week for three consecutive weeks. The persistence of the CAR-NK cells was detectable for up to 2 weeks after the last infusion. Notably, the infusion of CAR-NK cells resulted in the clearance of bone marrow (BM) disease as evident from BLI one week after the second infusion of CAR-NK cells while unengineered NK-cells failed to clear bone marrow disease. Assessment of CAR-NK cell efficacy in two different AML PDX models is ongoing, along with additional in vitro and in vivo studies that further demonstrate the ability of the SENTI-202 NOT EMCN Logic Gate to significantly protect healthy HSCs from off-tumor toxicity. In conclusion, CAR-NK cells expressing the SENTI-202 OR Gate and crIL15 demonstrate robust anti-tumor activity against primary AML cells within in vitro cytotoxicity assays and in vivo AML PDX models, including VEN-r AML. These promising preclinical results highlight the potential of SENTI-202 as a targeted and selective therapy for AML, offering a new avenue for overcoming the challenges posed by the heterogeneity of the AML proteomic landscape.

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

Abstract P6-11-15: Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which limited targeted therapies are available. Therefore, conventional chemotherapy remains the backbone of standard neoadjuvant treatment (NACT) for TNBC patients. Unfortunately, ~45% of patients will have substantial residual tumor burden post neoadjuvant chemotherapy, leading to poor prognoses (PMID: 28135148). Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is upregulated and is a therapeutic vulnerability in chemoresistant TNBC (PMID: 30996079; Baek et al., BioRxiv doi.org/10.1101/2022.02.25.481996). However, mechanisms driving increased oxphos in chemoresistant TNBC are not understood. Upregulated fatty acid (FA) metabolism is a common adaptation in tumors, providing an energy source through fatty acid β-oxidation (FAO), and promoting lipid accumulation after fatty acid synthesis (FAS) when energy needs are met. Chemotherapy can induce oxidative stress through the generation of reactive oxygen species. Cancer cells adapt to these damaging molecules by increasing de novo lipogenesis, resulting in the accumulation of lipid droplets (LDs) in the cytosol (PMID: 32782526, 20876798). We hypothesize that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC. Methods: Using orthotopic patient-derived xenograft (PDX) models of TNBC (PIM001-P, PMID: 30996079, HCI-010, PMID: 22019887; WHIM14, PMID:24055055), we are measuring protein levels of fatty acid synthase (FASN) in vehicle tumors vs residual tumors surviving treatment with the standard front-line neoadjuvant chemotherapy regimens (Adriamycin plus cyclophosphamide (AC), docetaxel, carboplatin, or docetaxel+carboplatin) using immunohistochemistry (IHC). Vectra 3 microscopy (Akoya) is being used to quantify tumor cell-specific staining. We complemented our IHC analysis with reverse-phase protein array (RPPA). To assess LD accumulation in residual PDX tumors, we conducted transmission electron microscopy (TEM). To complement these PDX studies, we modeled the residual tumor metabolic state in cultured human TNBC cells. Following treatment with the IC50 of standard chemotherapeutic agents (AC, carboplatin, paclitaxel, docetaxel), we assessed oxphos by measuring oxygen consumption rate (OCR) using a Seahorse Bioanalyzer (Agilent). Further, we tested LD accumulation using LipidTOX staining. In ongoing studies, we are measuring incorporation of 13C palmitate into the tricarboxylic acid cycle (TCA) prior to and following chemotherapy treatments to assess if lipids fuel mitochondrial metabolism in residual TNBC cells. Results/Discussion: IHC in the PIM001-P PDX model after in vivo AC treatment revealed increased levels of FASN in post-AC residual tumors compared to the treatment-naive tumors. Further, key proteins involved in fatty acid synthesis, FASN and Acetyl-CoA carboxylase, were significantly increased in residual PIM001-P cells that survived AC compared to vehicle by RPPA. TEM analysis of the HCI-010 PDX revealed significantly more LDs in carboplatin-treated tumors compared to vehicle. This finding was supported by increased LDs observed in TNBC cell lines treated with NACT compared to vehicle in our LipidTOX analyses. Taken together, these data indicate that NACT induces increased expression of key lipid metabolism proteins and accumulation of cytosolic LDs. Our future experiments will reveal if chemoresistant TNBC cells preferentially utilize and incorporate lipids into the tricarboxylic acid cycle, in turn driving oxphos. These data have the potential to provide rationale for the incorporation of FAO/LD inhibitors in sequential combinations with conventional chemotherapies to more effectively kill TNBC cells that are chemo-refractory. Citation Format: Katherine E. Pendleton, Mokryun L. Baek, Junegoo Lee, Lin Tan, Hannah L. Johnson, Lacey E. Dobrolecki, James P. Barrish, Michael T. Lewis, Philip L. Lorenzi, Fabio Stossi, Gloria V. Echeverria. Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-15.

Establishing Brain Tumor Stem Cell Culture from Patient Brain Tumors and Imaging Analysis of Patient-Derived Xenografts.

Brain tumor stem cells (BTSCs) have been isolated from different types of brain tumors including glioblastoma. Although BTSCs share common characteristics with neural stem cells (NSCs), such as capacity to self-renew and undergo long-term proliferation, they have tumor-propagating capabilities. A small population of BTSC can give rise to secondary tumor when transplanted into severe immunodeficient (SCID) mice. The histological and cytological features, as well as genetic heterogeneity of the xenografted tumors in mice, closely resemble those of primary tumors in patients. Patient-derived xenografts (PDX), therefore, provide a clinically relevant model to study brain tumors. Here, we describe our protocol for establishing BTSC cultures following surgical excision of human brain tumors and the procedures to conduct PDX studies in SCID mice. We also provide our detailed step-by-step protocol on in vivo imaging system (IVIS) of the PDX tumors as a noninvasive method to trace the cells and tumor volume.

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

The IL7R-Antagonist OSE-127 Blocks Acute Lymphoblastic Leukemia Development Via a Dual Mode of Action

Acute lymphoblastic leukemia (ALL) may arise from precursor B or T cells (BCP- and T-ALL, respectively) and treatment is based on polychemotherapy. There is an urgent need for novel immunotherapy targets to reduce therapy toxicity and to target relapsed/refractory (r/r) disease, especially in T-ALL where immunotherapy approaches are scarce. The Interleukin-7-receptor (IL7R) has been shown to play a pivotal role in the development of both BCP-ALL and T-ALL (Geron et al. 2022; Silva et al. 2022). The IL7Rα-chain (CD127)-targeting antibody OSE-127 has a good safety profile in healthy volunteers (NCT03980080) and has entered phase 2 trials in Ulcerative Colitis (NCT04882007) and Sjögren's syndrome (NCT04605978). We previously reported high preclinical efficacy of OSE-127 in BCP-ALL patient derived xenograft (PDX) models of different cytogenetic backgrounds (Lenk and Baccelli et al. ASH, 2021). However, the preclinical efficacy of OSE-127 in T-ALL had not yet been investigated and the mechanism of action underlying its anti-leukemic efficacy remained unclear. First, we investigated the in vivo efficacy of OSE-127 treatment on BCP-ALL and T-ALL cells by conducting a preclinical phase 2-like PDX study. Two NSG mice per patient were injected with BCP-ALL (n=13) or T-ALL (n=9) PDX cells (including 7 r/r ALL samples), randomly assigned into treatment groups and OSE-127 therapy was initiated when 1% leukemic blasts were detected in the peripheral blood (PB). OSE-127 treated mice showed a reduction of PB blasts in 21/22 (95%) PDX samples in vivo when the corresponding control mice showed clinical signs of overt leukemia (Figure 1A). This translated into a 1.6-fold prolongation of median survival (MS) of OSE-127 treated PDX mice (P<0.0001, Figure 1B). Of note, blast reduction strongly correlated with CD127 expression levels in the samples (Pearson r=0.5372, P=0.0099). Next, we characterized the mode of action of OSE-127. As expected, OSE-127 blocked STAT5 phosphorylation in IL7-dependent ALL cell lines and PDX samples. As OSE-127 is not internalized by CD127 expressing cells (Belarif et al. 2018), we investigated whether OSE-127 might also exert immune-mediated effects. We found that OSE-127 did not promote antibody-dependent cellular cytotoxicity (ADCC), but efficiently induced macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) in BCP- and T-ALL cell lines and PDX cells in vitro, in a manner dependent on CD127 expression levels (Pearson r=0.5000, P=0.0352, Figure 1C-D). In line with these observations, inactivating mutations of the Fc fraction of OSE-127 (OSE-127-LALAPG) prevented ADCP of ALL cells in vitro, while conserving its antagonistic properties on IL7R signaling. To further evaluate the contribution of ADCP to the anti-leukemic activity of OSE-127, we compared the in vivo efficacy of OSE-127 and OSE-127-LALAPG in ALL samples with dysfunctional IL7R signaling (e. g., DND41 cells showing constitutive pathway activation due to IL7Rα mutations or NALM6 cells being absent of P-STAT5 induction upon IL7 stimulation). In this context, OSE-127-LALAPG did not impact leukemic engraftment as compared to control treatment, while OSE-127 significantly reduced leukemic burden, thereby substantiating that ADCP contributes to the anti-leukemic efficacy of OSE-127 (Figure 1E). Finally, we conducted a phase2-like PDX overt leukemia study testing OSE-127 alone or in combination with Dexamethasone/Vincristine/PEG-Asparaginase chemotherapy employing PDX samples with high CD127 expression (6 BCP-ALL and 3 T-ALL-PDX samples with >50% CD127+ cells in flow cytometry analysis). OSE-127 alone significantly prolonged the survival of PDX mice in comparison to control treatment (MS 106d vs 79d, P=0.0071), which was comparable to the effect of chemotherapy (MS 106d vs 79d, P=0.0037). Importantly, the strongest effects were observed when combining OSE-127 with chemotherapy (MS 79d vs undefined, P=0.0003, Figure 1F). Overall, we propose OSE-127 as a novel potential immunotherapeutic agent for the treatment of BCP-ALL and T-ALL, particularly in r/r disease and in combination with chemotherapy. Due to its dual mode of action comprising ADCP induction and IL7R-pathway blockade, OSE-127 could be efficacious in CD127+ ALL with and without IL7R pathway alterations. L.L. and I.B. contributed equally to this work. N.P. and D.M.S. share senior authorship Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Direct Comparison of NSG and NBSGW Mice for MDS Patient-Derived Xenograft Models

Introduction: Patient-derived xenograft (PDX) models of Myelodysplastic Syndromes (MDS) based on conditioned NSG mice are the most commonly used and well-established. Recently, our group has published an extensive pre-clinical study including n=49 MDS PDX by employing such a model (Schmitt et al., Leukemia 2022). However, achieving adequate engraftment rates and sufficient numbers of PDX can still be challenging, especially for low risk MDS patient samples. Moreover, human erythropoiesis is known to be severely impaired in NSG mice due to residual macrophage activity. Thus, there is a need for further optimization of MDS PDX models. The KIT-deficient mouse strains NBSGW and NSGW41 have already been shown to provide improved engraftment of cord blood and support of human erythropoiesis compared to NSG mice. (McIntosh et al., Stem Cell Reports 2015; Rahmig et al., Stem Cell Reports 2016). Therefore, in this study, we compared NSG and NBSGW mice of both sexes regarding human MDS engraftment, lineage differentiation, clonal bone marrow (BM) composition and survival. Methods: BM-derived CD34+ hematopoietic stem cells and autologous in-vitro-expanded mesenchymal stroma cells from n=5 MDS patients (n=3 MDS-MLD, n=2 MDS-EB-2) with varying IPSS-R scores (n=1 very low, n=1 low, n=1 intermediate, n=2 high) were intrafemorally co-transplanted into male and female busulfan-conditioned NSG and non-conditioned NBSGW mice. As controls, samples of n=2 age-matched, healthy (HY) donors were used. Human long-term engraftment (>0.01 %) was determined 12 weeks post transplantation. Endpoint evaluation was performed after 19-30 weeks. Peripheral blood (PB) and BM were analyzed for different human immunophenotypic cell populations including CD33, CD34, CD41, CD45, CD71 and CD235a. Further, human CD45+ cells from PDX BM were isolated by FACS for DNA extraction. Subsequently, extracted PDX and original patient samples were subjected to NGS panel sequencing of MDS-specific mutations. Results: Overall, our data showed that female and male NBSGW mice enabled higher MDS engraftment rates than NSG mice. Engraftment (% human CD45) of MDS and HY samples in NBSGW females was found to be considerably higher in BM and PB than in NSG females (BM: 17.2% (0.01-71.1%) in NSG mice versus 43.7% (1.1-87.7%) in NBSGW; PB: 1.5% (0.01-6.4%) in NSG mice versus 3.5% (0.03-18.4%) in NBSGW). Moreover, NBSGW mice showed improved support of human thrombopoiesis, as well as higher levels of erythropoiesis and CD34+ cells in both MDS and HY PDX compared to NSG mice. However, survival of NSG females was significantly longer than for NBSGW females (p=0.0024), as well as for NSG (p=0.0006) and NBSGW (p<0.0001) males. Interestingly, males of both strains showed lower engraftment rates and shorter overall survival in the experiment as compared to females. Observed early deaths of males were not associated with engraftment rates. Panel sequencing results of n=4 MDS patients and their corresponding PDX showed no differences between NSG and NBSGW mice in mutational profiles and variant allele frequencies of transplanted MDS samples. Both strains were able to faithfully recapitulate the patient's clonal BM composition. Conclusions: Female NBSGW mice are superior engrafters for primary MDS samples in comparison to female NSG mice and provide the opportunity to investigate human thrombopoiesis and erythropoiesis in-vivo. While NSG PDX were already proven to faithfully recapitulate the clonal BM composition of transplanted patient samples, we found that the same is true for NBSGW mice. However, female NSG mice were more robust in terms of survival. This has to be taken into account when designing long-term PDX studies. Finally, in our study, male mice of both strains were shown to be inferior acceptors for PDX as compared to females.

S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer

BackgroundCDK4/6 inhibitors combined with endocrine therapy has become the preferred treatment approach for patients with estrogen receptor-positive metastatic breast cancer. However, the predictive biomarkers and mechanisms of innate resistance to CDK4/6 inhibitors remain largely unknown. We sought to elucidate the molecular hallmarks and therapeutically actionable features of patients with resistance to CDK4/6 inhibitors.MethodsA total of 36 patients received palbociclib and endocrine therapy were included in this study as the discovery cohort. Next-generation sequencing of circulating tumour DNA in these patients was performed to evaluate somatic alterations associated with innate resistance to palbociclib. Then the candidate biomarker was validated in another independent cohort of 104 patients and publicly available datasets. The resistance was verified in parental MCF-7 and T47D cells, as well as their derivatives with small interfering RNA transfection and lentivirus infection. The relevant mechanism was examined by RNA sequencing, chromatin immunoprecipitation and luciferase assay. Patient-derived organoid and patient-derived xenografts studies were utilized to evaluated the antitumor activity of rational combinations.ResultsIn the discovery cohort, S6K1 amplification (3/35, 9%) was identified as an important reason for innate resistance to CDK4/6 inhibitors. In the independent cohort, S6K1 was overexpressed in 15/104 (14%) patients. In those who had received palbociclib treatment, patients with high-expressed S6K1 had significantly worse progression free survival than those with low S6K1 expression (hazard ratio = 3.0, P = 0.0072). Meta-analysis of public data revealed that patients with S6K1 amplification accounted for 12% of breast cancers. Breast cancer patients with high S6K1 expression had significantly worse relapse-free survival (hazard ratio = 1.31, P < 0.0001). In breast cancer cells, S6K1 overexpression, caused by gene amplification, was sufficient to promote resistance to palbociclib. Mechanistically, S6K1 overexpression increased the expression levels of G1/S transition-related proteins and the phosphorylation of Rb, mainly through the activation of c-Myc pathway. Notably, this resistance could be abrogated by the addition of mTOR inhibitor, which blocked the upstream of S6K1, in vitro and in vivo.ConclusionsS6K1 amplification is an important mechanism of innate resistance to palbociclib in breast cancers. Breast cancers with S6K1 amplification could be considered for combinations of CDK4/6 and S6K1 antagonists.

Abstract 649: Targeting progesterone receptor (PR) with the antiprogestin onapristone in patient-derived xenograft (PDX) models of estrogen receptor positive (ER+), PR positive (PR+) bone metastasis of breast cancer

Abstract Background: Onapristone (ONA) is a progesterone receptor (PR) antagonist that prevents PR-mediated DNA transcription. ONA is currently being evaluated in metastatic breast cancers (MBC), as well as other hormone-dependent cancers. Recently, therapeutic options for metastatic estrogen receptor (ER) positive breast cancers have been expanded by the introduction of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), administered in combination with endocrine treatments, as first line therapy. Moreover, the selective PI3K inhibitor alpelisib (ALP) has been approved for the treatment of PIK3CA-mutated endocrine resistant MBC. The objective of this study was to evaluate the efficacy of ONA in combination with fulvestrant (FUL) and palbociclib (PAL) or ALP in different PDX models established from ER and PR positive breast cancers. Methods: PDX models were established from primary tumors or biopsies from bone metastases from endocrine therapy patients with progressing tumors. ER and PR expression were analyzed by immunohistochemistry (IHC) and western blot (WB) analysis in 17 PDX models. The anti-tumor activity of onapristone alone or combined with FUL and PAL was tested in 2 ER+/PR+ PDX studies, while the combination with ALP+FUL was tested in one PIK3CA-mutated PDX study. Phosphorylation of both PR and ER-PR interactions were analyzed in treated tumors by WB and by the in situ Proximity Ligation Assay (PLA), respectively. Results: PR expression was found in 1/9 PDX established from primary breast tumors and 4/8 PDX established from bone metastases. ONA in vivo activity was tested in 2 PDX of bone metastases: PDX BC1101 (PR low) and BC1117 (PR high). BC1101 showed amplification of FGFR1 and CCND1 genes, while BC1117 has an activating mutation of PIK3CA gene. In the low PR PDX BC1101, treatment with ONA was ineffective in both the monotherapy and combination setting. Conversely, in the PDX with high expression of PR, BC1117, ONA treatment, given as monotherapy, decreased tumor growth. The anti-tumor activity of ONA+FUL+PAL combination was significantly increased as compared to FUL+PAL, with the majority of xenografts showing tumor regression. Expression of PR and phospho-PR were inhibited in the tumors treated by FUL, ONA+FUL, and ONA+FUL+PAL. Interaction between ER and PR, analyzed by the in situ PLA assay, was inhibited in the ONA+FUL treated xenografts ±PAL. Finally, treatment with the triple combination of ONA+FUL+ALP was also highly effective and significantly greater than the combination of FUL+ALP. RNAseq analysis of treated xenografts is ongoing to identify transcriptomic changes in the different treatment arms. In conclusion, our study demonstrates that ONA improved the response to endocrine treatment using CDK4/6 or PI3K inhibitors in a MBC-derived PDX model with high PR expression. Citation Format: Martin Lehr, Ahmed Dahmani, Léa Huguet, Rania El-Botty, Charlène Thiebaut, Muriel Romancer, Paul Cottu, Elisabetta Marangoni. Targeting progesterone receptor (PR) with the antiprogestin onapristone in patient-derived xenograft (PDX) models of estrogen receptor positive (ER+), PR positive (PR+) bone metastasis of 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 649.

Abstract 1881: Drug combination screening of Ipatasertib and Abemaciclib with other targeted agents in complex multicellular tumor spheroids from the NCI-60 and the National Cancer Institute’s Patient-Derived Models Repository

Abstract Drug combinations are frequently used to improve clinical efficacy, to minimize toxicity, and to reduce the development of drug resistance. Here, we investigated the growth inhibitory activities of the pan-Akt inhibitor ipatasertib and the CDK4/6 inhibitor abemaciclib in combination with other targeted agents. Twelve well-characterized patient-derived cancer cell lines from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm) and seven established cell lines from the NCI-60 tumor cell line panel (https://dtp.cancer.gov/discovery_development/nci-60/cell_list.htm) were grown as multicellular 3D complex spheroids. The complex spheroids, a mixture of tumor cells (60%), endothelial cells (25%), and mesenchymal stem cells (15%), were grown for 3 days before drug(s) were added. All agents were tested both alone and in combinations at multiple concentrations up to their reported clinical Cmax value and cell viability was assayed using CellTiter-Glo 3D seven days after drug exposure. While abemaciclib had minimal activity as a single agent, ipatasertib was noticeably selective for tumor cells harboring activating PI3K/AKT/mTOR pathway variants. Dual inhibition of the PI3K/AKT/mTOR and RAS/MEK/ERK pathways was one of the most effective combinations. For example, the combination of ipatasertib with the MEK inhibitor selumetinib or the ERK inhibitor ravoxertinib resulted in additive and/or synergistic cytotoxicity in over half the complex spheroid models screened. The V600E variant-specific BRAF inhibitor vemurafenib and the KRAS G12C selective inhibitor sotorasib in combination with ipatasertib showed activity in the one BRAF V600E and two KRAS G12C variant containing complex spheroid models, respectively. Another effective ipatasertib combination was vertical inhibition of the PI3K/AKT/mTOR pathway with the mTORC1/2 kinase inhibitor sapanisertib, which demonstrated additive and/or synergistic responses across multiple complex spheroid models. For abemaciclib, the most successful combination was with the CDK2/7/9 inhibitor BMS-387032, which achieved greater than one log of cytotoxicity in the majority of the complex spheroid models. For the combination of abemaciclib and selumetinib, there was a high correlation between the responses of two patient-derived cell lines grown as complex spheroids and their corresponding patient-derived xenografts (PDX). Additional PDX studies are planned for promising drug combinations from this in vitro screen. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Rene Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Siddhartha Paul, Nathan P. Coussens, Ralph E. Parchment, Joel Morris, John Wright, Naoko Takebe, Beverly A. Teicher, James H. Doroshow. Drug combination screening of Ipatasertib and Abemaciclib with other targeted agents in complex multicellular tumor spheroids from the NCI-60 and the National Cancer Institute’s Patient-Derived Models Repository [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 1881.