Resistance In Preclinical Models Research Articles

Associations of dietary fructose with survival of patients (pts) with metastatic cancer of the urothelium (UC) and renal cell carcinoma (RCC) on immune checkpoint blockade (ICB).

4571 Background: The gut microbiome is a modifiable determinant of ICB efficacy. Dietary fiber is associated with longer progression-free survival (PFS) on ICB in melanoma and UC, putatively via effects on the gut microbiome. Other dietary factors may also impact ICB efficacy. Fructose promotes adverse health effects and induces ICB resistance in pre-clinical models via upregulation of the antiapoptotic enzyme heme oxygenase-1. Thus, we set out to explore effects of fructose intake on ICB efficacy. Methods: In a retrospective analysis of a prospectively collected cohort, we leveraged dietary data collected with the validated Harvard Willett Food Frequency Questionnaire from pts with advanced UC and RCC initiating ICB at Memorial Sloan Kettering to assess for associations between baseline dietary fructose and PFS and overall survival (OS). Tumor mutational burden (TMB) was estimated via MSK-IMPACT. Associations of fructose with clinical outcomes were evaluated using univariate (UV) and multivariable (MV) Cox proportional hazards regression. Fructose was normalized via log transformation and treated as a continuous variable. MV models adjusted for fiber intake and prior ICB exposure. UC models adjusted for TMB and Bellmunt risk score and RCC models for histology and IMDC score. Results: From 2/2021-6/2022, 88 eligible pts enrolled. Median follow-up was 10.2 and 17.0 months among pts with UC (n = 40) and RCC (n = 48), respectively; 32 RCC pts (67%) were IMDC intermediate/poor risk and 21 UC pts (52%) had visceral metastases. High fructose and fiber intake correlated (rho 0.66). Among UC pts, higher fructose was associated with shorter PFS after adjusting for covariables (Table). In the MV model, high fiber was associated with longer PFS (HR 0.2, 95% CI 0.07-0.62, p = .004). Among RCC pts, fructose was not associated with PFS. Fructose was associated with OS in the RCC UV model, but this association was not statistically significant in the MV model (Table). In an exploratory model for PFS pooling all pts, a test for interaction between fructose intake and disease type (UC vs RCC) showed p = .01. Conclusions: Dietary fructose is associated with shorter PFS in UC pts treated with ICB, but not RCC pts. Discrepant results between UC and RCC require further study and may be attributable to differences in therapy or cancer biology. Our findings provide insights into potentially beneficial dietary interventions. Efforts to characterize effects of fructose on the gut microbiome are ongoing. [Table: see text]

Liver and pancreatic-targeted interleukin-22 as a therapeutic for metabolic dysfunction-associated steatohepatitis

Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress β-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed. To maximise beneficial effects whilst reducing the risk of epithelial proliferation and cancer, we designed short-acting IL-22-bispecific biologic drugs that successfully targeted the liver and pancreas. Here we show 10-fold lower doses of these bispecific biologics exceed the beneficial effects of native IL-22 in multiple preclinical models of MASH, without off-target effects. Treatment restores glycemic control, markedly reduces hepatic steatosis, inflammation, and fibrogenesis. These short-acting IL-22-bispecific targeted biologics are a promising new therapeutic approach for MASH.

Abstract PO2-13-02: Detection of SPEN mutations in advanced breast cancer by circulating tumor cell-free DNA

Abstract Background: Alterations in the SPEN gene are rare in primary breast cancer (~3%, ref: TCGA). SPEN is a hormone-inducible transcriptional repressor with known functions in orchestrating X-inactivation in females. Additionally, SPEN has been implicated as a potential estrogen receptor co-repressor and molecular partner of NCOR2 as well as epigenetic modifiers such as KMT2D and HDACs via C-terminal interactions. SPEN loss-of-function is associated with tamoxifen resistance in preclinical models. However, the landscape of SPEN alterations in advanced breast cancer remains poorly described. The primary objective of this study was to evaluate the frequency and different types of SPEN alterations in metastatic breast cancer (MBC). Methods: A query was performed on all patients at an academic institution with available genomic testing from Guardant360, a next-generation sequencing cell-free DNA (cfDNA) assay, evaluating 500 genes in total. Patients with MBC were identified, and specific alterations in SPEN as well as co-alterations were extracted. Silent and germline mutations were excluded from analysis. Retrospective review was conducted to determine clinicopathologic characteristics such as age, hormone and HER2 receptor status, menopausal status, and therapy at time of the detected mutation. Results: Among 366 patients with MBC and available cfDNA, 8.5% (n=31) had an alteration detected in the SPEN gene. Median age of patients with SPEN mutations was 66 years (range 33-92). A majority of patients (90%) had HR+ disease (n=28/31), with HR-/HER2+ disease and TNBC additionally representing 3% (n=1/31) and 6% (n=2/31) of cases, respectively. SNVs were most frequent, comprising 89% (n=42/47) of all alterations. Indels were additionally identified in 11% (n=5/47), and no CNVs or fusions were noted. Nonsense and frameshift mutations represented 8/47 (17%) and 2/47 (4%) of all alterations respectively. No recurrent mutational hotspots were detected. The most common co-alterations in descending order were PIK3CA, TP53, ERBB2, ARID1A, MDM4, PPM1D, NF1, DNMT3A, ATM, and ESR1. Association between SPEN mutations and clinical outcomes will be presented at the meeting. Conclusions: SPEN mutations were detected mostly in patients with HR+/HER2- MBC and at a higher frequency than reported in primary disease via TCGA. SNVs were common, though it remains unclear whether these comprise driver versus passenger mutations. While no mutational hotspots were identified, several novel loss-of-function truncating mutations were identified, which warrant further validation of functional and clinical significance. SPEN alterations in metastatic breast cancer Alterations detected by cell-free DNA sequencing in patients with metastatic breast cancer and annotated by type of alteration and receptor subtype. Citation Format: Charles Dai, Haley Barnes, Arielle Medford, Annika Putur, Jennifer Keenan, Beverly Moy, Seth Wander, Ryan Corcoran, Aditya Bardia. Detection of SPEN mutations in advanced breast cancer by circulating tumor cell-free DNA [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-02.

Sonrotoclax overcomes BCL2 G101V mutation–induced venetoclax resistance in preclinical models of hematologic malignancy

AbstractVenetoclax, the first-generation inhibitor of the apoptosis regulator B-cell lymphoma 2 (BCL2), disrupts the interaction between BCL2 and proapoptotic proteins, promoting the apoptosis in malignant cells. Venetoclax is the mainstay of therapy for relapsed chronic lymphocytic leukemia and is under investigation in multiple clinical trials for the treatment of various cancers. Although venetoclax treatment can result in high rates of durable remission, relapse has been widely observed, indicating the emergence of drug resistance. The G101V mutation in BCL2 is frequently observed in patients who relapsed treated with venetoclax and sufficient to confer resistance to venetoclax by interfering with compound binding. Therefore, the development of next-generation BCL2 inhibitors to overcome drug resistance is urgently needed. In this study, we discovered that sonrotoclax, a potent and selective BCL2 inhibitor, demonstrates stronger cytotoxic activity in various hematologic cancer cells and more profound tumor growth inhibition in multiple hematologic tumor models than venetoclax. Notably, sonrotoclax effectively inhibits venetoclax-resistant BCL2 variants, such as G101V. The crystal structures of wild-type BCL2/BCL2 G101V in complex with sonrotoclax revealed that sonrotoclax adopts a novel binding mode within the P2 pocket of BCL2 and could explain why sonrotoclax maintains stronger potency than venetoclax against the G101V mutant. In summary, sonrotoclax emerges as a potential second-generation BCL2 inhibitor for the treatment of hematologic malignancies with the potential to overcome BCL2 mutation–induced venetoclax resistance. Sonrotoclax is currently under investigation in multiple clinical trials.

CPX-351 exploits the gut microbiota to promote mucosal barrier function, colonization resistance, and immune homeostasis

AbstractCPX-351, a liposomal combination of cytarabine plus daunorubicin, has been approved for the treatment of adults with newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes, because it improves survival and outcome of patients who received hematopoietic stem cell transplant compared with the continuous infusion of cytarabine plus daunorubicin (referred to as “7 + 3” combination). Because gut dysbiosis occurring in patients with AML during induction chemotherapy heavily affects the subsequent phases of therapy, we have assessed whether the superior activity of CPX-351 vs “7 + 3” combination in the real-life setting implicates an action on and by the intestinal microbiota. To this purpose, we have evaluated the impact of CPX-351 and “7 + 3” combination on mucosal barrier function, gut microbial composition and function, and antifungal colonization resistance in preclinical models of intestinal damage in vitro and in vivo and fecal microbiota transplantation. We found that CPX-351, at variance with “7 + 3” combination, protected from gut dysbiosis, mucosal damage, and gut morbidity while increasing antifungal resistance. Mechanistically, the protective effect of CPX-351 occurred through pathways involving both the host and the intestinal microbiota, namely via the activation of the aryl hydrocarbon receptor–interleukin-22 (IL-22)–IL-10 host pathway and the production of immunomodulatory metabolites by anaerobes. This study reveals how the gut microbiota may contribute to the good safety profile, with a low infection-related mortality, of CPX-351 and highlights how a better understanding of the host-microbiota dialogue may contribute to pave the way for precision medicine in AML.

Pharmacological inhibition of MYC to mitigate chemoresistance in preclinical models of squamous cell carcinoma.

Rationale: Cisplatin-based chemotherapy is the first-line treatment for late-stage head and neck squamous cell carcinoma (HNSCC). However, resistance to cisplatin has become a major obstacle for effective therapy. Cancer stem cells (CSCs) are critical for tumor initiation, growth, metastasis, and chemoresistance. How to effectively eliminate CSCs and overcome chemoresistance remains a key challenge. Herein, we confirmed that MYC plays critical roles in chemoresistance, and explored targeting MYC to overcome cisplatin resistance in preclinical models. Methods: The roles of MYC in HNSCC cisplatin resistance and cancer stemness were tested in vitro and in vivo. The combined therapeutic efficiency of MYC targeting using the small molecule MYC inhibitor MYCi975 and cisplatin was assessed in a 4‑nitroquinoline 1-oxide-induced model and in a patient-derived xenograft model. Results: MYC was highly-expressed in cisplatin-resistant HNSCC. Targeting MYC using MYCi975 eliminated CSCs, prevented metastasis, and overcame cisplatin resistance. MYCi975 also induced tumor cell-intrinsic immune responses, and promoted CD8+ T cell infiltration. Mechanistically, MYCi975 induced the DNA damage response and activated the cGAS-STING-IRF3 signaling pathway to increase CD8+ T cell-recruiting chemokines. Conclusions: Our findings suggested that targeting MYC might eliminate CSCs, prevent metastasis, and activate antitumor immunity to overcome cisplatin resistance in HNSCC.

Combination of BET Inhibition with Radiation Induces Anti-Tumor Immune Response in Pre-Clinical Models of ER-Positive Breast Cancer

Despite advances in multidisciplinary care, the risk of late recurrence in Estrogen Receptor-positive (ER+) breast cancer (BC) patients following standard-of-care treatment ranges from 20-52% at 20 years. While immune checkpoint inhibitors (CPI), have shown enormous potential in inducing long term remission in some cases, patients with ER+ BC show poor response to these agents. The objective of this study is to develop effective radiation therapy (RT)-based combination regimens that induce anti-tumor immunity against immunologically cold ER+ breast tumors. We have previously shown that pharmacological inhibition of bromodomain and extraterminal domain (BET) family of epigenetic regulator proteins overcomes both endocrine therapy resistance and radiation resistance in pre-clinical models of ER+ BC by reversing pathological reprogramming of transcriptional and DNA repair pathways, respectively. In this study, we explore the potential of RT+BET inhibition combination to elicit anti-tumor immunity against ER+ BC. A unique ER+ BC model, MXT+, was used to generate tumors in female, BDF1 mice. PD-L1 expression following RT or RT+OTX015 (a BET inhibitor) was assessed by Western blot analysis. Comet assay was used to assess the degree of unrepaired DNA damage following treatment with RT alone, OTX015 alone and RT+OTX015. Multiplex IHC was used to assess changes in tumor microenvironment after various treatments. Tumor growth characteristics were established following treatment with vehicle, RT alone (8 Gy x 3), OTX015 alone (100 mg/kg x 3 days) and RT+OTX015. Using murine ER+ BC model, MXT+, we show that RT is a potent inducer of PD-L1 expression. Combination of RT+OTX015 completely ablated RT-mediated PD-L1 induction at the transcription level. OTX015 also accentuated RT-induced DNA damage through inhibition of NHEJ pathway, resulting in increased micronuclei formation (which activate cGAS/STING pathway, a critical bridge between innate and adaptive immunity). Combination of OTX015 with RT also caused a statistically significant (on unpaired t test) increase tumor infiltrating lymphocytes. In in vivo therapeutic studies, combination of OTX015 with RT (8 Gy x 3) resulted in extraordinary synergy and caused complete inhibition of long-term tumor growth. Treatment with RT alone (8 Gy x 3) or OTX015 alone (on 3 consecutive days) only caused modest inhibition of tumor growth. ANOVA with Dunnett's test was used to adjust for multiple comparisons and showed statistically significant difference in tumor growth between OTX015+RT arm and OTX alone as well as OTX015+RT arm and RT alone. Patients with ER+ BC face high risk of late recurrence after definitive treatment and show poor response to immune CPIs. We show that combination of BET inhibition with RT induces anti-tumor immunity and completely prevents tumor growth. Clinical validation of this regimen is warranted to prevent late recurrences in ER+ BC patients.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

BackgroundMetabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker.MethodsBioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats.ResultsMiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells.ConclusionsMiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

Phase II study of biweekly TAS-102, irinotecan and bevacizumab in pre-treated metastatic colorectal cancer (TABAsCO).

3590 Background: The efficacy of 2nd line FOLFIRI plus VEGF inhibition (VEGFi) is limited for metastatic colorectal cancer (mCRC). TAS-102 is a novel oral anti-metabolite approved for pts with mCRC following standard treatment with FP, oxaliplatin and irinotecan. TAS-102 has a distinct mechanism of action from 5-FU and overcomes 5-FU resistance in preclinical models, and in the clinic. We hypothesized that the combination of TAS-102 with irinotecan (TAS-IRI) and bevacizumab (BEV) would prove superior to FOLFIRI/VEGFi. Methods: Phase II, single-arm study of TAS-IRI and BEV in 2nd-line mCRC. Eligible patients had received prior treatment with a fluoropyrimidine and oxaliplatin in the advanced setting or experienced recurrence within 12 mos of this regimen in the adjuvant setting. Adequate organ function, ECOG PS 0-1, measurable disease (RECIST 1.1), no prior irinotecan or TAS-102 exposure and candidacy for BEV were required. Pts were treated with BEV 5 mg/kg IV, irinotecan 180 mg/m2 IV (day 1 and 15), and TAS-102 25 mg/m2 orally bid (days 2-6 and 16-20) in 4-week cycles (PMID: 31924737). Treatment continued until disease progression or unacceptable toxicity. The primary endpoint was median progression-free survival (PFS). Assuming a median PFS for historic control (FOLFIRI/VEGFi) of 6 mos vs. 9 mos for TAS-IRI plus BEV (HR: 0.67), treatment of 36 evaluable pts would achieve 80.5% power (at α = 0.1) to detect such an effect. Results: Forty-nine patients were enrolled; 42 were eligible and 38 had at least one disease assessment (median age 59 yr, 47% male, 74% colon primary, tumor KRAS/NRAS mutated in 55%/21%). The median PFS was 8.7 mos (range: 6.7-11.8 mos, p = 0.009), meeting the primary endpoint. The most common reason for study treatment discontinuation was progressive disease (53%). The objective response rate was 16% (90% CI: 8.0-27.3%); the median OS was 16.5 mo (range: 11.8-25.3). Twenty-eight pts (67%) had a treatment-related adverse event (TRAE) G3 or higher (Table). The most common TRAEs were gastrointestinal and hematologic. Grade 3/4 neutropenia occurred in 34%, with only 1 episode of febile neutropenia, though G-CSF use was frequently needed to maintain dose density/intensity. Conclusions: TAS-IRI plus BEV is an effective 2nd line therapy for patients with mCRC. The median PFS appears higher compared to historical controls (FOLFIRI/VEGFi). Neutropenia is common and can affect dose density/intensity mandating use of G-CSF. A randomized study versus standard of care therapy is warranted. (Clinical trial registration ClinicalTrials.gov NCT04109924). Clinical trial information: NCT04109924 . [Table: see text]

UCLA l-08: A phase ib/II study of combined HER inhibition adding necitumumab and trastuzumab to osimertinib in patients with refractory EGFR-mutated lung cancer.

e21165 Background: Patients (pts) with metastatic EGFR-mutated NSCLC who have progressed on osimertinib have limited treatment options. Dual EGFR with HER2 blockade results in complete and long-term reversal of osimertinib resistance in preclinical models. Methods: This is an investigator-initiated, single arm, open label phase 1b/2 study to identify the recommended phase 2 dose (RP2D), safety, tolerability, and preliminary efficacy of the combination of osimertinib, necitumumab, and trastuzumab (ONT) in adults with histologically confirmed, metastatic NSCLC with an activating and sensitizing EGFR mutation who have progressed on osimertinib as their most recent treatment. Study pts receive daily oral osimertinib in conjunction with necitumumab and trastuzumab intravenously every other week. In phase 1b, a 3+3 dose-escalation design is used to determine the RP2D (Table 1). In phase 2, if ≥ 2 responses are seen out of 10 pts treated at the RP2D, an additional 10 pts will be accrued. Patient reported outcomes (PRO-CTCAE) and quality of life (FACT-L) data are collected prospectively. Efficacy is assessed as objective response rate (ORR) based on RECIST 1.1 criteria. Results: 22 pts (median age = 63; range = 40-82) have been enrolled and treated. The most commonly observed treatment-related AEs seen at any DL included acneiform rash (63.6%), headache (45.5%), nausea (40.9%), dry skin (36.4%), diarrhea (31.8%), paronychia (27.3%), oral mucositis (27.3%), vomiting (22.7%), fatigue (18.2%), chills (18.2%), and anorexia (18.2%). Grade 3 treatment-related toxicities were acneiform rash (22.7%), diarrhea (4.5%), decreased lymphocyte count (4.5%) and hypertension (4.5%). There were no treatment-related Grade ≥ 4 AEs. 12 evaluable pts were enrolled in phase 1b [3 at Dose Level (DL) 1, 3 at DL 2, and 6 at DL 3]; 3 non-evaluable pts were replaced. Pts treated at DL 1 and DL 2 had no dose-limiting toxicities (DLTs) and 1/6 pts at DL 3 had a DLT (persistent grade 3 rash). Due to grade 2 and 3 rash and paronychiae occurring after cycle 1 at DL 3, DL2 was determined to be the RP2D.The proportion of evaluable phase 1b pts achieving partial response or stable disease was 58.3% (7/12). Seven pts have been enrolled at DL2. Conclusions: ONT toxicities are manageable, and the combination has promising preliminary efficacy in refractory EGFR-mutated metastatic NSCLC. Evaluation of phase 2, stage 1 is expected to be completed by April 2023 and will be reported at the meeting. Clinical trial information: NCT04285671 . [Table: see text]

Iruplinalkib (WX‑0593), a novel ALK/ROS1 inhibitor, overcomes crizotinib resistance in preclinical models for non-small cell lung cancer

Despite remarkable initial responses of anaplastic lymphoma kinase (ALK) inhibitors in ALK-positive non-small cell lung cancer (NSCLC) patients, cancers eventually develop resistance within one to two years. This study aimed to compare the properties of iruplinalkib (WX‑0593) with other ALK inhibitors and report the comprehensive characterization of iruplinalkib against the crizotinib resistance. The inhibitory effect of iruplinalkib on kinase activity was detected. A kinase screen was performed to evaluate the selectivity of iruplinalkib. The effect of iruplinalkib on related signal transduction pathways of ALK and c-ros oncogene 1 (ROS1) kinases was examined. The cellular and in vivo activities of ALK inhibitors were compared in engineered cancer-derived cell lines and in mice xenograft models, respectively. Human hepatocytes derived from three donors were used for evaluating hepatic enzyme inducing activity. HEK293 cell lines expressing transportors were used to invesigated the drug interaction potential mediated by several transporters. The results showed iruplinalkib potently inhibited the tyrosine autophosphorylation of wild-type ALK, ALKL1196M, ALKC1156Y and epidermal growth factor receptor (EGFR)L858R/T790M. The inhibitory effects of iruplinalkib in patient-derived xenograft and cell line-derived xenograft models were observed. Moreover, iruplinalkib showed robust antitumor effects in BALB/c nude mice xenograft models with ALK-/ROS1-positive tumors implanted subcutaneously, and the tumor suppressive effects in crizotinib-resistant model was significantly better than that of brigatinib. Iruplinalkib did not induce CYP1A2, CYP2B6 and CYP3A4 at therapeutic concentration, and was also a strong inhibitor of MATE1 and MATE2K transporters, as well as P-gp and BCRP. In conclusion, iruplinalkib, a highly active and selective ALK/ROS1 inhibitor, exhibited strong antitumor effects in vitro and in crizotinib-resistant models.

Abstract P3-11-03: Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer

Abstract Endocrine receptor-positive (ER+) breast cancers (BC) comprise over 70% of all breast cancers and are the leading cause of BC-related deaths in women. Despite available targeted therapies against ER+ BC, recurrence arises primarily due to the development of endocrine therapy resistance and metastases. Thus, there is an unmet need to identify novel biomarkers for treating ER+ BC patients with metastases. We have identified a neuroimmune molecule, Semaphorin 7a (SEMA7A), as a potential biomarker for endocrine therapy resistance, increased relapse and decreased overall survival in ER+ BC patients. We have also found that SEMA7A promotes tumor growth, angio- and lymphangiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis and endocrine therapy resistance in pre-clinical models. Specifically, we have shown that SEMA7A+ ER+ MCF7 tumors result in lung metastases that are resistant to the endocrine therapy, fulvestrant, in part, via downregulation of ER in vivo – posing the need to identify novel, druggable targets for SEMA7A+ ER+ BC. SEMA7A is a membrane-bound protein that we have shown can inhibit tumor cell death via integrin-mediated activation of PI3K/Akt pro-survival signaling. Thus, we hypothesized that that SEMA7A+ ER+ BC cells may be sensitive to PI3K inhibition in combination with fulvestrant. Our results demonstrate that SEMA7A+ MCF7 cells are sensitive to the combination of fulvestrant and PI3K inhibition by alpelisib, which is FDA approved for ER+ BC patients with activating mutations in PI3K; this combination also inhibited tumor cell growth and decreased tumor sphere formation. In complementary studies, we identified that Singleminded 2s (SIM2s), which is expressed in breast epithelial cells and inhibits EMT, is downregulated SEMA7A+ MCF7 tumors. Additionally, our studies show that while ER-mediated signaling turns on SEMA7A expression prolonged expression of SEMA7A results in downregulation of ER, and the loss of SIM2s causes downregulation of ER expression and upregulation of SEMA7A in MCF7 cells. Further, while SEMA7A-overexpressing (OE) tumor cells exhibit increased tumor-promoting PI3K/Akt survival signaling, we found that SIM2s plays a role in suppression of PI3K/Akt survival signaling. Therefore, we are investigating the interactions between SIM2s, SEMA7A and the survival signaling pathways involved in their crosstalk. Our recent results show that MCF7 SIM2-knockout (KO) cells exhibit increased SEMA7A promoter activity, SEMA7A expression, activation of Akt signaling, EMT phenotypes and decreased ER expression. Also, addition of exogenous SEMA7A protein reduces SIM2s expression and promoter activity. Since SIM2s correlates with inhibition of PI3K/Akt signaling and decreased SEMA7A, and SEMA7A promotes survival signaling, we are investigating additional therapeutic strategies to prevent endocrine therapy resistance via direct inhibition of SEMA7A and/or restoration of SIM2s, including inhibition of COX-2 and Akt signaling. Citation Format: Rachel Steinmetz, Garhett Wyatt, Traci Lyons, Weston Porter, Lyndsey Crump. Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-03.

Abstract P4-08-16: Selective Androgen Receptor Modulators in combination with CDK4/6 inhibitors demonstrate anti-cancer activity in preclinical treatment resistant ER+AR+ breast cancer models

Abstract Background: The Androgen Receptor (AR) is expressed in up to 90% of all ER+ breast cancers and has been associated with better patient outcome. While androgens were used at a high dose as an anticancer therapy historically, this was discontinued with the advent of Tamoxifen due to virilising effects. Non-steroidal, tissue selective AR modulators (SARMs) represent an attractive alternative, offering a targeted approach to AR activation. Recent compelling pre-clinical data has established that the AR is a tumour suppressor in ER+ breast cancers and that AR activation with a natural androgen or a SARM suppressed ER-driven tumour growth, in preclinical models of endocrine-sensitive and -resistant ER+ breast cancer. Here, we evaluate the efficacy of a SARM (enobosarm) and a natural AR ligand (dihydrotestosterone, DHT) in the context of metastatic, CDK4/6 inhibitor (CDK4/6i) resistant breast cancer. Methods: Enobosarm and palbociclib treatments were evaluated in vitro by colony forming assays using CDK4/6i resistant (MCF7 PalbR) and both endocrine and CDK4/6i resistant (MCF7 cTamPalbR) cell lines. Next, enobosarm or DHT and palbociclib treatment were evaluated in vivo using endocrine and CDK4/6i resistant ER+ patient derived xenograft models (PDX) and cell line xenograft models. IHC, RNA and ChIP sequencing (AR, ER, H3K27ac) were subsequently performed on the harvested tumours. Results: While in vitro and in vivo growth of CDK4/6i resistant preclinical models was inhibited by treatment with a SARM or DHT alone, growth inhibition was more potent and durable in combination with a CDK4/6i. Gene set enrichment analysis of RNA-seq data integrated with ChIP-seq data revealed upregulation of an AR gene signature associated with a better prognosis following treatment with SARM. Co-treatment with a SARM and a CDK4/6i also enhanced AR signalling compared to SARM alone indicating an interaction of the two signalling pathways. Conclusion: Our data indicates that combination treatment with an AR agonist and a CDK4/6i represents a novel therapeutic strategy for CDK4/6i resistant ER+AR+ breast cancers Citation Format: Allegra Freelander, Geraldine laven-Law, Leila Eshraghi, Nimmy Geetha, Peta Somerville, Marie Pickering, Sarah Alexandrou, C. Elizabeth Caldon, Wayne D. Tilley, Theresa E. Hickey, Elgene Lim. Selective Androgen Receptor Modulators in combination with CDK4/6 inhibitors demonstrate anti-cancer activity in preclinical treatment resistant ER+AR+ breast cancer models. [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 P4-08-16.

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

Despite approval of B-cell lymphoma (BCL)-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and mouse double minute 2 (MDM2) could overcome venetoclax resistance in preclinical models. Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115). The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein. Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

160 (PB040) - The AXL inhibitor, TP-0903, reverses EMT and shows activity in non-small cell lung cancer preclinical models

Background: Adenocarinomas frequently adopt mesenchymal properties to become metastatic, aggressive and drug-resistant. Non-small cell lung cancer (NSCLC), a leading cause of cancer-related deaths, is a well-understood cancer type to utilize an epithelial-to-mesenchymal transition (EMT) to acquire drug resistance in preclinical models. This is particularly applicable in resistance to EGFR inhibitors (e.g. osimertinib) in EGFR+ NSCLC, where resistance has been linked to EMT and increased AXL expression, a known driver of the mesenchymal phenotype.

MA07.05 Phase 1b/2 Study of Combined HER Inhibition in Refractory EGFR-mutated Metastatic Non-small Cell Lung Cancer (NSCLC)

Patients with metastatic EGFR-mutated NSCLC who have progressed on osimertinib have limited treatment options. Dual EGFR blockade with HER2 blockade has been shown to have complete and long-term reversal of osimertinib resistance in preclinical models.

Abstract 2991: Survival reliability of Glioblastoma on CD38

Abstract Glioblastoma (GBM) is a fatal malignancy of the brain. Unfortunately, the current multimodality management approach does not lead to long term survival benefits. Several prognostic factors such as Blood Brain Barrier (BBB) & Blood Tumor Barrier (BTB), Glioma Stem-like Cells (GSCs), an immunosuppressive tumor microenvironment (TME) and genetic heterogeneity promotes GBM progression. To improve treatment efficacy, these factors should be addressed in a rational manner. Our pre-clinical investigations indicate that maintenance of CD38 expression, a type II transmembrane glycoprotein, is vital to GBM survival. CD38 could be an ideal biologically relevant target as it is homogeneously expressed on tumor cells and its TME elements. Furthermore, accessibility of FDA approved anti-CD38 therapies, non-overlapping toxicities and potential to cross the BBB has granted approval of our phase I study using Radiation/Temozolomide and Immunotherapy with Daratumumab to Improve Antitumor Efficacy in newly diagnosed Glioblastoma (PRIDE) (NCT04922723). In brief, we performed Western blot (WB), RT-qPCR, and flow cytometry to examine CD38 expression on human GBM cell lines and microglia (LN229, T98G, DI-321, SA21 and HMC3) including CD38 transfected cells. We used trans well assays to analyze changes in CD38 expression in several environments. MTS viability assay was used to study the viability of Temozolomide on GBM cells. Basal expression of CD38 on GBM cells was assessed, showing that CD38 expression varies amongst cell lines and components of its TME. CD38 negative (flow sorted) cells regain CD38 expression around Day 4 after sorting, indicating that CD38 is crucial for GBM survival. We then sorted cells into CD38 negative and positive and performed trans well experiments with overexpressing and knock down cells, revealing that CD38 expression changes based upon surrounding cells in its environment. Cells transfected with shRNA against CD38 have a slower proliferative rate compared to wild-type and CD38 overexpressing cells. We also saw that LN229 Temozolomide (TMZ) resistant (LN229-TR) cells also have a slower proliferative rate and show an increase in CD38 expression. We also observed that CD133, SOX2 and Nestin are significantly over expressed in LN229-TR compared to LN229 CD38 WT, while sharing similar CD38 expression as GSCs and malignant GBM cells. IC50 of TMZ on CD38 overexpressing cells was significantly higher than CD38 WT GBM cells suggesting that CD38 expression contributes towards treatment resistance. All experiments were performed in triplicate and p values less than 0.05 were considered significant. Our preliminary data suggests that CD38 is regulating the proliferation rate as well as stem cell like features of GBM cells. Moreover, overexpression of CD38 is leading to TMZ resistance in pre-clinical models. Our ongoing in vitro and in vivo gene deletion experiments will further solidify the biological importance of CD38 in GBM. Citation Format: Leland Earp, Srishti Kala, Sonikpreet Aulakh. Survival reliability of Glioblastoma on CD38 [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 2991.

Abstract PD2-02: Combination CDK4/6 inhibition and AR agonism suppresses the growth of CDK4/6 inhibitor resistant breast cancers

Abstract Resistance to standard-of care-therapies is a significant clinical challenge in estrogen receptor positive (ER+) breast cancer. Cyclin dependent kinase 4/6 inhibitors (CDK4/6i) in combination with endocrine therapies (ET) is the current standard-of-care for advanced metastatic ER+ breast cancer; however, resistance to this combination is considered inevitable, leading to disease progression. The androgen receptor (AR) is expressed in up to 90% of all ER+ breast cancers, and has been associated with better patient outcome. Compelling recent pre-clinical data demonstrates that selective androgen receptor AR modulators (SARMs) act to suppress ER-driven tumour growth of endocrine-sensitive and -resistant models of ER+ breast cancer. Furthermore, a recent clinical trial evaluating the efficacy of SARMs has shown clinical benefit in patients with ER+/AR+ metastatic breast cancer (NCT02463032). We hypothesise that the SARMS, either alone or in combination with a CDK4/6i, would be an effective treatment for tumours that are sensitive and resistant to ET and CDK4/6i. We report therapeutic efficacy of combination AR agonism + CDK4/6i in CDK4/6-naïve and -resistant pre-clinical models, including cell lines and patient derived xenograft (PDX) models. We demonstrate that combination SARM + CDK4/6i potently and durably inhibited in vitro and in vivo tumour growth. Additionally, we provide evidence that in vivo treatment with the CDK4/6i Palbociclib increased AR expression and signalling, highlighting an interaction of the two signalling pathways not previously described. In conclusion, our data provides a pre-clinical rationale for combination SARM + CDK4/6i in CDK4/6i resistant ER+ breast cancer. Citation Format: Allegra Freelander, Geraldine Laven-Law, Leila Eshraghi, Kee Ming Chia, Marie Pickering, Aliza Yong, Ashleigh Wilkinson, Sarah Alexandrou, C. Elizabeth Caldon, Theresa E Hickey, Wayne D Tilley, Elgene Lim. Combination CDK4/6 inhibition and AR agonism suppresses the growth of CDK4/6 inhibitor resistant breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-02.

Venetoclax in Acute Myeloid Leukemia: Molecular Basis, Evidences for Preclinical and Clinical Efficacy and Strategies to Target Resistance.

Simple SummaryVenetoclax is a BH3-mimetics that specifically inhibits the antiapoptotic protein BCL-2. The drug has considerably expanded the treatment options of acute myeloid leukemia (AML) patients unfit for intensive treatment regimens. In recent phase III trials, combination of venetoclax with the hypomethylating agents azacitidine or with low dose cytarabine, was associated with a 48–65% response rate and a 7.2–14.7 months overall survival. Recent studies also suggest that the drug is highly efficient in combination with high-dose chemotherapy and targeted therapeutics, including IDH and FLT3 inhibitors. In this review, we summarize the molecular basis of BCL-2 inhibition in AML, the effect of venetoclax in preclinical models and clinical studies and discuss the main molecular pathways and therapeutic options for overcoming venetoclax resistance. Identifying new therapeutic strategies increasing BH3 mimetics activity and/or sensitizing toward apoptotic cell death will represent a major challenge for treating AML patients in the near future.Venetoclax is a BH3-mimetics agent specifically interacting with the antiapoptotic protein BCL-2, facilitating cytochrome c release from mitochondria, subsequent caspases activation, and cell death. Utilization of venetoclax has profoundly changed the landscape of treatment for the poor-prognosis category of AML patients unfit for intensive chemotherapy. In the phase III VIALE-A study, Venetoclax, in combination with the hypomethylating agent azacitidine, showed a 65% overall response rate and 14.7-month overall survival, in comparison with 22% and 8 months in the control arm. These results led to the widespread use of venetoclax in this indication. Other combination regimens, consisting of low-intensity, intensive, or targeted therapies are currently under evaluation. Despite promising results, preventing relapses or resistance to venetoclax is still an unmet clinical need. Numerous studies have been conducted to identify and overcome venetoclax resistance in preclinical models or in clinical trials, including the inhibition of other antiapoptotic proteins, the induction of proapoptotic BH3-only proteins, and/or the targeting of the mitochondrial metabolism and machinery.

Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.