Activity In Preclinical Models Research Articles

Phase Ib/II study of XmAb23104 (PD1 X ICOS) and XmAb22841 (CTLA-4 X LAG3) combination in metastatic melanoma refractory to prior immune checkpoint inhibitor therapy with and without CNS disease.

TPS9595 Background: Immune checkpoint inhibitors (ICI) have greatly improved outcomes for patients by targeting programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte–associated antigen 4 (CTLA4) and Lymphocyte Activation Gene-3 (LAG-3) to increase immune-mediated anti-tumor response. Despite these advances, ~50% of patients with metastatic melanoma do not respond to standard of care ICI. We strive to address this unmet clinical need through a Phase Ib/II, first-in-human, multi-center trial of two bispecific antibodies targeting four different immune checkpoints: XmAb23104 (PD1 X ICOS) and XmAb22841 (CTLA-4 X LAG3) in patients with melanoma refractory to ICI. This is the first trial of quadruple checkpoint inhibitor therapy. Furthermore, bispecific antibodies may allow simultaneous checkpoint blockade on the same cell and have been shown to heighten T cell activation in pre-clinical models. Two ongoing phase I trials of these bispecific antibodies with PD1 therapy have observed an immune related adverse event (irAE) rate of 20-24% with efficacy analysis ongoing (NCT03752398; NCT03849469). We postulate that treatment with XmAb23104 and XmAb22841 will enhance immune activation, overcome resistance, and improve disease control for patients refractory to traditional ICI. Methods: Eligible patients must have advanced/metastatic melanoma and have progressed on prior PD1/PD-L1 inhibitor or PD1/CTLA4 dual inhibition. Cutaneous, acral, mucosal and melanoma of unknown origin are allowed. Patient with CNS metastasis are allowed if these are asymptomatic and stable on MRI imaging obtained within 4 weeks of enrollment. Patients with prior unresolved irAE or irAE ≥ grade 3 are not allowed. XmAb23104 and XmAb22841 will be given on D1 and D15 of a 28-day cycle for 4 cycles, after which XmAb23104 (PD1 X ICOS) will be given alone for a total of 2 years. The phase I portion will be a dose escalation using standard 3+3 design of XmAb22841 (CTLA-4 X LAG3) with three dose levels (0.3 mg/kg, 1 mg/kg and 3 mg/kg). The dose of XmAb23104 (PD1 X ICOS) will remain at 10 mg/kg. These doses were selected based on results of prior early phase trials when these agents were combined with PD1. Once the P2RD of XmAb22841 (CTLA-4 X LAG3) is established, we will move on to dose escalation using a Simon’s 2-stage design. This portion will include two arms: Arm A consisting of 17 patients without CNS disease and Arm B consisting of 17 patients with CNS disease (total 34). Primary endpoints include dose limiting toxicities (DLT)/irAE and Objective Response Rate (ORR) at the time of best response by RECIST 1.1. Secondary endpoints include progression-free survival, overall survival, CNS response. At the time of this abstract submission, Cohort 1 of dose escalation has begun with the first few patients still within the DLT observation window. Clinical trial information: NCT05695898 .

OPEN‐LABEL PHASE 1/2 STUDY OF CC‐99282, A CEREBLON E3 LIGASE MODULATOR (CELMOD) AGENT ± RITUXIMAB, IN PATIENTS WITH RELAPSED/REFRACTORY (R/R) NON‐HODGKIN LYMPHOMA (NHL)

Introduction: CC-99282 is a novel, oral, small molecule CELMoD® agent that co-opts cereblon to induce targeted degradation of Ikaros/Aiolos, transcription factors critical for B-cell malignancy development. Compared with immunomodulatory drugs (IMiD), CC-99282 had 10- to 100-fold enhanced antiproliferative and apoptotic activity in preclinical models of diffuse large B-cell lymphoma (DLBCL), while maintaining immunostimulatory effects. We previously reported promising efficacy and a predictable and manageable safety profile of CC-99282 in patients (pts) with R/R NHL (Michot et al. EHA, 2022). Methods: CC-99282-NHL-001 (NCT03930953) is a 2-part, multicentre, first-in-human study with dose escalation of CC-99282 monotherapy (part A) and expansion ± combination partners (part B) in pts with R/R DLBCL or follicular lymphoma (FL) who progressed after ≥2 lines of therapy, or pts with R/R DLBCL with ≥1 line of standard therapy and transplant-ineligible. Part A dosing is CC-99282 0.2–0.8 mg QD on 3 intermittent schedules of 28 d cycles. Part B dosing is CC-99282 0.2 or 0.4 mg alone on 2 intermittent schedules or with rituximab (RTX). Here, we report updated efficacy results for CC-99282 monotherapy (part A) and new safety data for CC-99282 + RTX (part B). Results: For part A, as of Sep 14, 2022, 50 pts were treated (38 DLBCL, 12 FL; median age 66 y); 3 pts (6%) completed, 7 (14%) were ongoing and 40 (80%) discontinued, most commonly due to progressive disease (n = 33, 66%). Median # of prior systemic anticancer therapies was 3 (range 1–8). For doses ≥0.4 mg on 7/14 or 14/28 d schedules, the overall response rate was 43% (17/40 evaluable pts; 10/30 DLBCL, 7/10 FL), with complete response in 7 and partial response in 10 pts. Responders included pts previously treated with CAR T cell therapy and/or IMiD/CELMoD agents. Responses were durable (Figure), with median duration of response (mDOR) of 299 d (range 48–898; median follow-up [mFU] 293) in pts with DLBCL and mDOR of 448 d (135–930; mFU 572) in pts with FL. For part B, as part of the safety run-in, 12 pts were treated with CC-99282 + RTX (9 DLBCL, 3 FL; median age 61y). With mFU of 6 wks (range 4–28), neutropenia was the most common any-grade treatment-emergent adverse event (AE), occurring in 5/12 (42%) pts, including 25% (3/12) grade 3/4 neutropenia. No febrile neutropenia occurred. There were no dose reductions, discontinuations, or dose-limiting toxicities (DLTs). Severe AEs included fever (n = 2), and dyspnoea and fatigue (n = 1 each). Similar safety profiles were observed at both CC-99282 dose levels, 0.2 and 0.4 mg, combined with RTX. The research was funded by: Celgene, a Bristol-Myers Squibb Company Keywords: aggressive B-cell non-Hodgkin lymphoma, combination therapies, indolent non-Hodgkin lymphoma Conflicts of interests pertinent to the abstract J. M. Michot Honoraria: Ideogen Educational grants: Amgen J. Chavez Consultant or advisory role: Advisory board of Kite and Novartis C. Carpio Consultant or advisory role: Consultancy: Takeda, Regeneron, Novartis Educational grants: Celgene E. Bachy Consultant or advisory role: Consultancy: Roche, Takeda, Incyte Honoraria: Kite/Gilead, Novartis Research funding: Daiishi F. Morschhauser Consultant or advisory role: Consultancy: Roche, Gilead, Genmab, Novartis, Abbvie J. M. Jørgensen Consultant or advisory role: Advisory board: Roche, Gilead, Novartis, BMS R. Cordoba Consultant or advisory role: Advisory role: BMS, Janssen, Abbvie, Astra Zeneca, Eli Lilly, Beigene, Kite P. Bories Consultant or advisory role: Consultancy: Abbvie; Advisory role: Kite/Gilead Research funding: BMS Educational grants: Kite/Giliead P. Patah Employment or leadership position: Employment: BMS Other remuneration: Equity ownership: BMS B. de Moucheron Employment or leadership position: Employment: BMS Stock ownership: BMS S. Carrancio Employment or leadership position: Employment: BMS Stock ownership: BMS C. Guarinos Employment or leadership position: Employment: BMS X. Zheng Employment or leadership position: Employment: BMS C. Mei Employment or leadership position: Employment: BMS M. Pourdehnad Employment or leadership position: BMS Stock ownership: BMS L. Nastoupil Honoraria: DC Therapeutics, Abbvie, BMS/Celgene, Incyte, Caribou Biosciences, Epizyme, Genentech, Gilead/Kite, Janssen, Novartis, Pfizer, Takeda Research funding: BMS/Celgene, Caribou Biosciences, Epizyme, Genentech, Gilead/Kite, IGM Biosciences, Janssen, Novartis, Pfizer, Takeda

First-in-human, phase I study of KC1036, a multiple kinase inhibitor, as a single agent in patients with advanced solid tumors.

e15000 Background: KC1036 is a novel and selective multi-kinase inhibitor that targets cell proliferation, angiogenesis, metastasis and drug resistance by inhibiting receptor tyrosine kinases (AXLs and VEGFRs) involved in the tumor microenvironment. KC1036 has demonstrated anti-tumor activity in preclinical models of solid tumors, including non-small cell lung cancer (NSCLC). Methods: This phase I, first-in-human study of KC1036 (NCT04387916) enrolled adult patients (pts) with advanced or metastatic solid tumors. This study was comprised of dose escalation (Part A: With the accelerated titration of the 10 mg QD, in which only one patient was enrolled, a standard 3+3 design was applied for 20, 40, 60, and 80 mg QD) followed by dose expansion (Part B) and recommended phase II dose (RP2D) expansion (Part C). The primary safety endpoints were to determine dose limiting toxicities (DLTs) and RP2D. Secondary endpoints included pharmacokinetics (PK) and preliminary efficacy evaluated per RECIST v1.1 criterion. Results: As of November 30, 2022, a total of 41 pts had received at least 1 dose of KC1036 orally with 13 pts in Part A (10 mg [n = 1], 20 mg [n = 3], 40 mg [n = 3], 60 mg [n = 4], 80 mg [n = 3]) and 28 pts in Part B (60 mg [n = 21], 80 mg [n = 7]). In Part A, no DLTs were observed, and the maximum tolerated dose (MTD) has not been reached. Consequently, 60 mg and 80 mg were recommended for Part B. Pts most commonly had NSCLC (31.7%, 13/41), esophageal cancer (14.6%, 6/41), colorectal cancer (12.1%, 5/41). All pts were heavily pre-treated with 65% (27/41) receiving three or more lines of therapy, and average targeted tumor size was 58.7 mm with the largest 162 mm. Of the 41 pts, the median duration of KC1036 exposure was 105.0 days (range, 51.0-168.0). Any-grade TRAEs occurred in 39 pts (95.1%), with 16 pts (39.0%) reported with ≥ Grade 3. The most commonly reported ≥ Grade 3 TRAEs at 60 mg was hypertension (16.7%, 4/24), and that at 80 mg was diarrhea (30.0%, 3/10). PK analysis showed that KC1036 exhibited with dose-proportional increases in exposure over the range of 10 to 40 mg, but increased less proportionally within the range of 40-80 mg. Of 36 pts who had at least one efficacy evaluation, 5 (13.9%) had a best response of partial response (PR) and 24 (66.7%) had stable disease (SD). Thus, ORR was 13.9% (95% CI: 4.7, 29.5) and DCR was 80.6% (95% CI: 64.0, 91.2). Among the 9 evaluable pts with lung adenocarcinoma (6 on 60 mg and 3 on 80 mg), 2 pts had PR, 5 had SD and 2 had progressive disease (PD) as best response. Sustainable PR for 42 weeks was observed in 1 patient with lung adenocarcinoma on 60 mg. Conclusions: KC1036 demonstrated a manageable safety profile and preliminary antitumor activity in pts with advanced solid tumors. Taking safety, efficacy and clinical PK into consideration, 60 mg QD was recommended as RP2D for Part C, and this part was ongoing. Further studies in pts with lung adenocarcinoma would be considered promising. Clinical trial information: NCT04387916 .

Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-γ is a major determinant of CD4+ CAR T-cell antitumor activity

CD4+ T cells and CD4+ chimeric antigen receptor (CAR) T cells display highly variable antitumor activity in preclinical models and in patients; however, the mechanisms dictating how and when CD4+ T cells promote tumor regression are incompletely understood. With the help of functional intravital imaging, we report that interferon (IFN)-γ production but not perforin-mediated cytotoxicity was the dominant mechanism for tumor elimination by anti-CD19 CD4+ CAR T cells. Mechanistically, mouse or human CD4+ CAR T-cell-derived IFN-γ diffused extensively to act on tumor cells at distance selectively killing tumors sensitive to cytokine-induced apoptosis, including antigen-negative variants. In anti-CD19 CAR T-cell-treated patients exhibiting elevated CAR CD4:CD8 ratios, strong induction of serum IFN-γ was associated with increased survival. We propose that the sensitivity of tumor cells to the pro-apoptotic activity of IFN-γ is a major determinant of CD4+ CAR T-cell efficacy and may be considered to guide the use of CD4+ T cells during immunotherapy.

The roles and implications of RNA m6A modification in cancer.

N6-Methyladenosine (m6A), the most prevalent internal modification in eukaryotic mRNA, has been extensively and increasingly studied over the past decade. Dysregulation of RNA m6A modification and its associated machinery, including writers, erasers and readers, is frequently observed in various cancer types, and the dysregulation profiles might serve as diagnostic, prognostic and/or predictive biomarkers. Dysregulated m6A modifiers have been shown to function as oncoproteins or tumour suppressors with essential roles in cancer initiation, progression, metastasis, metabolism, therapy resistance and immune evasion as well as in cancer stem cell self-renewal and the tumour microenvironment, highlighting the therapeutic potential of targeting the dysregulated m6A machinery for cancer treatment. In this Review, we discuss the mechanisms by which m6A modifiers determine the fate of target RNAs and thereby influence protein expression, molecular pathways and cell phenotypes. We also describe the state-of-the-art methodologies for mapping global m6A epitranscriptomes in cancer. We further summarize discoveries regarding the dysregulation of m6A modifiers and modifications in cancer, their pathological roles, and the underlying molecular mechanisms. Finally, we discuss m6A-related prognostic and predictive molecular biomarkers in cancer as well as the development of small-molecule inhibitors targeting oncogenic m6A modifiers and their activity in preclinical models.

202P A phase Ib/II study of OP-1250, an oral complete estrogen receptor (ER) antagonist (CERAN) and selective ER degrader (SERD) with palbociclib in patients (pts) with advanced or metastatic ER-positive, HER2-negative breast cancer (MBC)

OP-1250 is small molecule CERAN/SERD that binds to and completely blocks transcriptional activity of wild-type and mutant ER. OP-1250 was well tolerated in a phase I/II monotherapy study (OP-1250-001), and the recommended phase II dose is 120 mg once a day (qd). OP-1250 with palbociclib showed synergistic activity in preclinical models. Here we report updates of pharmacokinetics (PK), drug-drug interactions (DDI), safety and efficacy from a study of OP-1250 with palbociclib (OP-1250-002). Pts with advanced or MBC with progression on or after ≤1 line of endocrine therapy (prior CDK4/6 inhibitors and chemotherapy were allowed) were enrolled into sequential cohorts to receive escalating doses of OP-1250 PO qd with palbociclib 125 mg PO qd for 21 of 28 days, using a 3+3 design, followed by dose expansion. As of January 23, 2023, 20 pts have been treated with palbociclib and OP-1250 doses of 30/60/90/120 mg (n=3/3/3/11). Fourteen received prior CDK4/6 inhibitor; 11 received prior palbociclib. No DLTs occurred. The most common (≥4 pts) treatment emergent adverse events (AEs) were neutropenia, nausea, vomiting, anemia, gastroesophageal reflux, constipation, and thrombocytopenia (all were Grade 1-2, except neutropenia). Grade 3 neutropenia occurred in 11 pts (55%). No Grade 4 AEs occurred. The exposure of OP-1250 (n=18) was consistent with the monotherapy study. Palbociclib exposure at steady state was comparable to published monotherapy data when combined with OP-1250 at all dose levels tested. Anti-tumor activity has been observed including partial responses. OP-1250 did not affect palbociclib PK and no DDIs have been observed with this combination. OP-1250 and palbociclib combination was well tolerated, safety was consistent with individual profiles of each drug as a monotherapy. Tumor responses were observed in this heavily pretreated population. Expanding on our previous report (SABCS 2022), these data provide rationale to continue exploring OP-1250 with the approved dose of palbociclib. Updated data will be presented. (NCT0526610).

Available Systemic Treatments and Emerging Therapies for Breast Cancer Brain Metastases

In 2023, breast cancer brain metastases (BCBrM) remain a major clinical challenge gaining well-deserved attention. Historically managed with local therapies alone, systemic therapies including small molecule inhibitors and antibody-drug conjugates (ADCs) have shown unprecedented activity in recent trials including patients with brain metastases. These advancements stem from efforts to include patients with stable and active BCBrM in early- and late-phase trial design. Tucatinib added to trastuzumab and capecitabine improves intracranial and extracranial progression-free survival and overall survival in stable and active human epidermal growth factor receptor 2 (HER2+)-positive brain metastases. Trastuzumab deruxtecan (T-DXd) has both shown impressive intracranial activity in stable and active HER2+ BCBrMs challenging historical thinking of ADCs' inability to penetrate the central nervous system (CNS). T-DXd has shown potent activity in HER2-low (immunohistochemistry scores of 1+ or 2+, non-amplified by fluorescence in situ hybridization) metastatic breast cancer and will be studied in HER2-low BCBrM as well. Novel endocrine therapies including oral selective estrogen downregulators (SERDs) and complete estrogen receptor antagonists (CERANs) are being studied in hormone receptor-positive BCBrM clinical trials due to robust intracranial activity in preclinical models. Triple-negative breast cancer (TNBC) brain metastases continue to portend the worst prognosis of all subtypes. Clinical trials leading to the approval of immune checkpoint inhibitors have enrolled few BCBrM patients leading to a lack of understanding of immunotherapies contribution in this subgroup. Data surrounding the use of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors in patients with germline BRCA mutation carriers with CNS disease is hopeful. ADCs including those targeting low-level HER2 expression and TROP2 are under active investigation in triple-negative BCBrMs.

Abstract LB318: SB-4826, a first-in-class oral, covalent inhibitor of SUMO E1 that induces IFN signaling and inhibits tumor growth as monotherapy and in combination with immune checkpoint blockade

Abstract Sumoylation is a reversible post-translational modification of proteins by small ubiquitin-like modifier (SUMO) that regulates protein function and contributes to cellular response to stress. Sumoylation is an essential pathway for all cells and is involved in diverse processes such as inflammation, DNA damage response, signaling and cell survival/apoptosis. Due to its involvement in such cellular functions, sumoylation intersects with the majority of the hallmarks of cancer. Many cancers display elevated expression of SUMO pathway components, a mechanism co-opted by tumors to survive under stressful conditions. In addition to its tumor intrinsic functions, sumoylation has emerged as a novel target for activating anti-tumor immunity due to its role in regulating type I interferon (IFN) signaling. Targeting sumoylation is a compelling anti-cancer strategy due to the multiple mechanisms of action that may drive anti-tumor activity for cancer patients. We report the discovery of SB-4826, a novel, orally active, covalent small molecule inhibitor of the Sumo Activating Enzyme (SUMO E1), the initiating enzyme of the sumoylation cascade. SB-4826 selectively and irreversibly binds an allosteric pocket of SUMO E1 and drives potent biochemical and cellular activity. SB-4826 inhibits global sumoylation in cells and blocks proliferation across a broad panel (100+) of cancer cell lines. Cysteine-proteome profiling by mass spectrometry indicated that Cys-30 of SUMO E1 was the only cysteine-containing peptide (out of 14,541 measured) that met the criteria for covalent engagement indicating exquisite selectivity across the cellular proteome. In an in vivo pharmacodynamic model measuring IFN signaling SB-4826 treatment led to dose- and time-dependent induction of IFN regulated cytokines. SB-4826 treatment resulted in significant tumor growth inhibition in multiple in vivo models. In a syngeneic A20 lymphoma model SB-4826 led to tumor stasis. In a human RAJI xenograft model, SB-4826 induced near-complete inhibition of tumor growth as a single agent and resulted in tumor regression when combined with rituximab. In a mouse CT-26 colorectal tumor model which is generally refractory to anti-PD-1 treatment, SB-4826 significantly inhibited tumor growth as monotherapy and led to 60% complete tumor responses when combined with anti-PD-1. SB-4826 caused increased CD8+ T cell infiltration in CT-26 tumors supporting the role of SB-4826 in inducing IFN signaling and leading to a tumor microenvironment that is more responsive to immune checkpoint inhibition. SUMO E1 is a clinically validated cancer target as evidenced by recent data showing encouraging responses to TAK-981, an IV-administered, ATP-competitive inhibitor. SB-4826 is a differentiated and optimized SUMO E1 inhibitor that is administered orally and demonstrated improved activity in preclinical models. SB-4826 has favorable properties for development, is a new and promising cancer therapy, and is entering phase 1 clinical studies in 2023. Citation Format: Jude R. Canon, Ashley Callinan, Sarah Bradley, Laurie Wang, Mackenzie Kui, Ariel Wein, Gabriele Sulli, Peter Buchowiecki, Fang-Tsao Hong, Miles Kubota, Mary Walton, Victor J. Cee. SB-4826, a first-in-class oral, covalent inhibitor of SUMO E1 that induces IFN signaling and inhibits tumor growth as monotherapy and in combination with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB318.

Abstract CT272: Pharmacodynamic and immunophenotyping analyses of ATR inhibitor M1774 in a Phase I study in patients with solid tumors (DDRiver Solid Tumors 301)

Abstract Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase plays a critical role in the DNA damage response. M1774, a potent, selective, orally administered ATR inhibitor with antitumor activity in preclinical models, was evaluated in Part A1 of an open-label, single-arm study (NCT04170153) for safety, tolerability, maximum tolerated dose, pharmacokinetics (PK) and pharmacodynamics (PD). M1774 monotherapy in patients with advanced solid tumors was well-tolerated and the totality of evidence, including quantitative model-based analyses, suggested the recommended dose for expansion (RDE) as 180 mg QD 2 weeks on/1 week off.1 Here, we report findings of the M1774 PD and immunophenotyping analyses. Methods: M1774 PD was explored by assessing phosphorylation by ATR of CHK1 (p-CHK1) in tumor and of H2AX (γ-H2AX) in serial blood samples, stimulated ex vivo with the radiomimetic 4-Nitroquinoline N-oxide or Dimethyl sulfoxide as control. A flow cytometry quantitative assay was used to measure γ-H2AX in the CD45+ lymphocytes fraction. The effect of M1774 on the immunophenotype was explored by flow cytometry. Blood samples were collected at baseline, 3 and 24 hours after first M1774 administration on day 1 of cycle 1 for the γ-H2AX analysis, and on Days 1 and 15 of Cycles 1 and 2 before treatment for immunophenotyping. Results: Preclinical tumor tissue-blood bridging PD analyses in a mouse model demonstrated that the inhibition of γ-H2AX in lymphocytes highly correlated with inhibition of p-CHK1 in tumor. Clinical data of γ-H2AX levels and immunophenotyping were generated for the blood samples collected from the 55 participants of Part A1 of the study. Exploratory PK-PD analysis using γ-H2AX levels 3 h post-dose on day 1 across the doses predicted target inhibition >80% for doses ≥130 mg, suggesting target engagement. The levels of ɣ-H2AX at 24 h after first dose intake were variable and mean levels rebounded to baseline value. M1774 treatment did not cause any significant and consistent change in the levels of all explored immune cell subsets at the tested dose levels, including myeloid-derived suppressor cells, T and B lymphocytes, monocytes, and natural killer cells. Conclusions: PD analyses showed that M1774 efficiently inhibited ATR at the RDE without impacting the immunophenotype. 1TA Yap, et al. Ann Oncol. 2022; 33(suppl_7): S197-S224. Citation Format: Ruth Plummer, Anthony W. Tolcher, Timothy A. Yap, Giuseppe Sessa, Jatinder K. Mukker, Annick Seithel-Keuth, Christine Hicking, Zoltan Szucs, Ioannis Gounaris, Giuseppe Locatelli, Johann S. de Bono. Pharmacodynamic and immunophenotyping analyses of ATR inhibitor M1774 in a Phase I study in patients with solid tumors (DDRiver Solid Tumors 301) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT272.

Preclinical discovery and initial clinical data of WVT078, a BCMA × CD3 bispecific antibody.

B-cell maturation antigen (BCMA) is an ideal target in multiple myeloma (MM) due to highly specific expression in malignant plasma cells. BCMA-directed therapies including antibody drug conjugates, chimeric antigen receptor-T cells and bispecific antibodies (BsAbs) have shown high response rates in MM. WVT078 is an anti-BCMA× anti-CD3 BsAb that binds to BCMA with subnanomolar-affinity. It was selected based on potent T cell activation and anti-MM activity in preclinical models with favorable tolerability in cynomolgus monkey. In the ongoing first-in-human phase I dose-escalation study (NCT04123418), 33 patients received intravenous WVT078 once weekly at escalated dosing. At the active doses of 48-250 µg/kg tested to date (n = 26), the overall response rate (ORR) was 38.5% (90% CI: 22.6-56.4%) and the complete response rate (CRR, stringent complete response + complete response) was 11.5%, (90% CI: 3.2-27.2%). At the highest dose level tested, the ORR was 75% (3 of 4 patients). 26 (78.8%) patients reported at least one Grade ≥3 AE and 16 of these AEs were suspected to be drug related. 20 patients (60.6%) experienced cytokine release syndrome. WVT078 has an acceptable safety profile and shows preliminary evidence of clinical activity at doses tested to date.

Abstract 6253: SP-3164, a novel molecular glue degrader with activity in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) makes up approximately 10% of all hematologic malignancies, with increasing incidence worldwide. Though treatment options for newly diagnosed patients have evolved in recent years, they are not curative, and relapse is common with 2,500-3,000 relapsed or refractory (R/R) MM US patients seeking novel treatments, annually. Cereblon (CRBN)-binding Ikaros and Aiolos (I/A) protein degraders (i.e., lenalidomide (LEN) and pomalidomide (POM)) have had great success in treating MM. However, due to the unstable chiral nature of the CRBN-binding moiety, common to these molecules, many exist as racemic mixtures (1:1 mixture of interconverting (R)- and (S)-enantiomers). A growing body of evidence suggests that the (S)- enantiomer is the active anticancer species, while the (R)-enantiomer is inactive and potentially results in undesired activity. The ability to stabilize the active (S)- enantiomer represents a potential strategy to improve therapeutic outcomes. We are developing SP-3164, the deuterium-stabilized (S)-enantiomer of avadomide (AVA), an I/A-degrading molecular glue which has been studied in over 400 patients, demonstrating safety and activity in hematologic diseases. Our in vitro data demonstrate that SP-3164 more potently and rapidly degrades the cancer-promoting transcription factors, I/A, compared to LEN, POM, and AVA, while the stabilized (R)-enantiomer, SP-3165, neither binds CRBN nor degrades I/A. In preliminary in vivo studies, SP-3164 elicited significant tumor growth inhibition (TGI). Here, we investigate the activity of SP-3164 in MM, compared to established treatment regimens. SP-3164’s potential to degrade Aiolos in MM.1S cells was compared to SP-3165, AVA, and POM. SP-3164 rapidly and robustly degraded Aiolos in a time- and dose-dependent manner, more potently than AVA and POM, while SP-3165 demonstrated minimal Aiolos degradation. To investigate the effect of SP-3164 on cell survival, we treated NCI-H929 cells with SP-3164 (0.1 µM, 1 µM, and 10 µM) and SP-3165 (1 µM) and assessed apoptosis by flow cytometry. SP-3164 induced dose-dependent early and late apoptosis after 72 hours, while SP-3165 showed no induction of apoptosis. SP-3164’s in vivo activity was assessed and compared to SP-3165, LEN, and POM in an NCI-H929 mouse xenograft model. SP-3164 showed significant TGI as a single agent while SP-3165 seemed to support tumor growth. LEN and POM demonstrated minimal TGI. When combined with dexamethasone (DEX), SP-3164 had improved TGI compared to the combination of POM+DEX. These preclinical data demonstrate that SP-3164 is a potent CRBN-binding molecular glue with the ability to rapidly degrade cancer-promoting transcription factors and induce apoptosis in vitro. In vivo, SP-3164 has superior single agent and combination activity compared to approved molecular glues. These data support the continued development as a novel therapy in MM. Citation Format: Aundrietta D. Duncan, Daniela Y. Santiesteban, Nadeem Q. Mirza, Steve Horrigan, Sheila DeWitt, Vincent Jacques, P. Leif Bergsagel. SP-3164, a novel molecular glue degrader with activity in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6253.

Abstract 6320: CUSP06/AMT-707, a new CDH6-targeting antibody-drug conjugate, demonstrates potent antitumor activity in preclinical models

Abstract Cadherin-6 (CDH6), also known as K-cadherin, is a type II classic cadherin molecule that plays an important role in the embryonic development of the kidney, but has very limited expression in adult tissues. It is overexpressed in several human malignancies, primarily in ovarian, renal, but also (to a lesser degree) gastric, thyroid, cholangiocarcinoma and other tumor types. The characteristics of limited expression in normal tissues, high expression in tumor tissues, and rapid internalization upon antibody binding makes CDH6 a well-suited Antibody-drug Conjugate (ADC) target. We developed a novel CDH6-targeting ADC, CUSP06, consisting of a proprietary humanized antibody selective for CDH6, a protease cleavable linker, and an exatecan payload, with a drug-to-antibody ratio (DAR) of 8. CUSP06 selectively bound to cell surface CDH6 and was efficiently internalized into CDH6 positive ovarian and renal cancer cells. CUSP06 exhibited strong antiproliferative activity against several CDH6 positive cancer cell lines in vitro. Furthermore, compared to a DXd-based ADC, a exatecan-based ADC demonstrated improved bystander effect. Treatment with CUSP06 resulted in tumor regression in several CDH6 positive cell line derived xenograft (CDX) models, including PA-1, OVCAR3, and 786-O. In addition, CUSP06 demonstrated potent antitumor activity with tumor regression observed in two CDH6 low and high expressing patient derived xenograft (PDX) models. The preclinical activity of CUSP06 against CDH6-expressing tumors provide compelling support for the clinical development of CUSP06 in CDH6-expressing human cancers. CUSP06 is currently in IND-enabling studies. CUSP06 showed an expected toxicity profile consistent with it's exatecan payload in the ongoing pilot toxicology studies. We plan to initiate a Phase 1 first-in-human clinical trial in the 2nd half of 2023. Citation Format: Wei Lu, Jing Shi, Shu-Hui Liu, Nicole Covino, Xun Meng, Eric D. Slosberg. CUSP06/AMT-707, a new CDH6-targeting antibody-drug conjugate, demonstrates potent antitumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6320.

Abstract 6185: Debio 0123 is a selective WEE1 inhibitor that effectively penetrates the brain and demonstrates anti-tumor activity in preclinical models of glioblastoma

Abstract Background: Glioblastoma (GBM) is one of the most aggressive and hard-to-treat cancers with a 5-year survival rate of 5.8%, in part due to the presence of the blood brain barrier (BBB) that prevents most therapeutics from reaching the tumors at efficacious concentrations. Debio 0123 is a WEE1 inhibitor that has previously shown significant preclinical efficacy in combination with DNA damaging agents or radiotherapy and is currently being investigated in phase 1 clinical studies either as a monotherapy or in combination with carboplatin. Herein we present data that demonstrates Debio 0123 effectively penetrates in to the brain and subsequently leads to anti-tumor activity in GBM models both in vitro and in vivo as a monotherapy or in combination with standard-of-care therapies (SOC). Methods: Radio- and chemo-sensitizing effects of Debio 0123 were evaluated in GBM cell lines in vitro using standard clonogenic assays or cytotoxicity assays. In vivo, distribution and bioanalysis studies were performed to determine the amount of Debio 0123 present in the different parts of the brain and intracranially implanted tumors. Efficacy of Debio 0123 as monotherapy or in combination with the SOC, temozolomide (TMZ), was further assessed in vivo in U87MG-luc intracranial or subcutaneous xenograft models. Results: In vitro, Debio 0123 in combination with radiation enhanced cell death in GBM cell lines and reduced TMZ IC50 in primary GBM cell lines. In vivo studies demonstrated substantial Debio 0123 brain penetration in mice, rats and monkeys, with mean brain-to-plasma concentration ratios of ~0.6 and 1.52 and 4, respectively. Furthermore, a mean tumor-to-plasma ratio of 0.62 was determined in orthotopically implanted brain tumors in mice. In efficacy studies, Debio 0123 administered at 30 or 60mg/kg every day for 28 days displayed significant anti-tumor activity in a GBM xenograft model U87-MG implanted in immunodeficient mice either subcutaneously, with up to 57.5% tumor growth inhibition, or orthotopically in the brain, with up to 73.7% tumor growth inhibition. Finally, efficacy of Debio 0123 in combination with TMZ was also assessed in intracranially transplanted U87-MG tumors. Here, TMZ was highly active, however, the combination of Debio 0123 and TMZ resulted in significantly enhanced anti-tumor activity leading to sustained complete regressions in 75% of animals for up to 100 days and was well tolerated throughout the 28-day treatment. Conclusion: These results demonstrate that following oral administration, Debio 0123 effectively crosses the BBB, has significant efficacy as a monotherapy and improves response to standard-of-care chemo or radiotherapy in preclinical models. Collectively, these data highlight Debio 0123 as a promising candidate for further clinical development in GBM patients that may provide improved response to SOC therapy. Citation Format: Luke Piggott, Noemie Luong, Frederic Massiere, Annett Kunze, Christophe Chardonnens, Anne Vaslin. Debio 0123 is a selective WEE1 inhibitor that effectively penetrates the brain and demonstrates anti-tumor activity in preclinical models of glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6185.

Abstract 1661: Heart-sparing ALK5 inhibitor for treatment of gastrointestinal and liver cancers

Abstract Transforming growth factor beta (TGFβ) signaling has been implicated in many cancers and fibrotic diseases. Recent pharmacological intervention has shown promising activities in preclinical models and early clinical trials. There are over 15 investigational agents targeting this pathway currently in the clinic, but none have been approved. In particular, small molecule TGFβ inhibitors targeting ALK5 (TGFβ type 1 receptor) have been limited by mechanism-based heart valve toxicity to low dose and intermittent dosing regimens. NEX002 is an oral liver-targeted ALK5 inhibitor with low heart exposure for the treatment of liver cancer and other gastrointestinal cancers with liver metastases. The compound was designed following drug design principles from known liver-specific drugs, such as the statins. In animals, NEX002 exerts preferential distribution (pharmacokinetic) and target inhibition (pharmacodynamic) in liver over the heart. NEX002 has demonstrated activity in several intrahepatic and subcutaneous tumor models, and significantly enhanced the durability of anti-tumor efficacy in combination with lenvatinib, a VEGFR inhibitor approved for the treatment of hepatocellular carcinoma. No heart valve toxicity was observed when benchmarked against galunisertib (systemic ALK5 inhibitor) in toxicity studies with repeated daily dosing. These data suggest that our liver-targeted/heart-sparing approach may allow more robust target engagement compared to systemic ALK5 inhibitors through higher dose or more frequent dosing. Citation Format: Andrew T. Miller, Qihui Jin, David Plouffe, Manny Corpuz, Evelyn Rodrigo, Tom Y.- Wu. Heart-sparing ALK5 inhibitor for treatment of gastrointestinal and liver cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1661.

Abstract 1629: Prostate cancer RIPTAC™ therapeutics demonstrate activity in preclinical models of Enzalutamide-resistant prostate cancer

Abstract Background: Novel drugs are needed to tackle forms of prostate cancer that demonstrate resistance to hormonal agents. Halda has invented an innovative cancer treatment approach that does not rely on oncogenic drivers. Regulated Induced Proximity Targeting Chimera (RIPTAC™) therapeutics are heterobifunctional small molecules that work via a hold and kill mechanism that has the potential to overcome drug resistance mechanisms. RIPTAC therapeutics function by holding together two proteins, a cancer-specific protein, and a protein with essential function (EP) in a ternary complex, resulting in abrogation of the essential function and subsequent cancer cell death. We exemplify this platform to treat metastatic castration resistant prostate cancer (mCRPC), where the RIPTAC therapeutic utilizes the Androgen Receptor (AR) as a tumor specific protein to selectively inhibit an essential protein involved in transcriptional regulation and provide in vivo efficacy coupled with a therapeutic index. Methods: RIPTACs therapeutics were assayed for their ability to form a ternary complex with AR and the EP using a novel TR-FRET based assay in VCaP prostate cancer cells that harbor AR amplification. Selective apoptosis in ARhigh cells was observed using a Caspase 3/7 Glo assay (Promega). EP pharmacodynamic modulation was ascertained using qRT-PCR and western blotting in both in vitro and in vivo samples. RIPTACs were optimized for oral bioavailability, and tumor ternary complex formation in prostate cancer cell line-derived xenograft models.Results: RIPTAC therapeutics display nanomolar in vitro potency in AR:RIPTAC:EP ternary complex formation, which results in abrogation of the EP function and antiproliferative activity in prostate cancer cell lines, but not in AR-knockout control cells. The prostate cancer RIPTAC therapeutics are active in vitro against clinically relevant AR mutants. Lead molecules utilizing AR as a tumor specific protein are orally bioavailable in multiple preclinical species and induce ternary complex formation in VCaP tumor xenografts grown in mice. We present in vivo data where lead RIPTACs demonstrate significant tumor growth inhibition in several prostate cancer models and induce tumor regressions in VCaP xenografts grown in castrated male mice. Conclusions: Taken together, our in vitro mechanistic data and in vivo PD/efficacy observations in multiple prostate cancer models support further investigation of prostate cancer RIPTAC therapeutics as a novel heterobifunctional therapeutic modality in mCRPC. Citation Format: Xinheng Yu, Kyle J. Eastman, Kanak Raina, Kelli M. Jones, Chris D. Forbes, Abigail Hundt, Marco Garcia, Rebecca Stronk, Katia Howard, Andrew McGovern, Rebekka Chenard, Allison Denny, Mia Forgione, Kyle Bassoli, Ethan Garvin, James J. Mousseau, Hao Li, Madeline P. King, Amit Bhardwaj, Katherine J. Kayser-Bricker, Craig M. Crews. Prostate cancer RIPTAC™ therapeutics demonstrate activity in preclinical models of Enzalutamide-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1629.

Abstract 5991: Development and characterization of a novel CDK4/6 inhibitor for the treatment of CDK4/6 dependent cancers

Abstract CDK4/6 inhibitors give clinical benefit in the treatment of metastatic ER+/HER2- breast cancer (mBC) in combination with endocrine therapy. Although this class of inhibitor has been extensively studied in several other cancer types, CDK4/6 inhibitors have not been approved as a treatment for outside of ER+/HER2- cancer. We evaluated the clinically approved CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib—in a 500+ cell line screening platform and identified several cancers outside of breast that may have subpopulations that are sensitive to CDK4/6 inhibitors. Kinome and KiNativ analyses helped identify differences in sensitivity/selectivity that we observed between each CDK4/6 inhibitor. Comprehensive molecular profiling of the cell lines at baseline allowed us to screen for potential associations with sensitivity to these compounds. These analyses identified potential biomarkers of sensitivity in non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) that will later be evaluated in human clinical studies. Having thoroughly studied the CDK4/6 class and discovered new opportunities for clinical development, we set out to develop a CDK4/6 inhibitor that would have the best properties of the two distinguished class members. Using our proprietary chemistry, we developed UCT-03-008, a potent CDK4/6 small molecular inhibitor with a pharmacology known to enhance therapeutic benefit in mBC. Pharmacokinetic properties of UCT-03-008 are improved over those of other CDK4/6 inhibitors. UCT-03-008 has activity in preclinical models as a monotherapy and yields in vivo efficacy in multiple cancer models outside of ER+/HER2- breast cancer, including CRC and NSCLC. In summary, UCT-03-008 is a CDK4/6 inhibitor with optimized pharmacological properties. Clinical development of this compound will be guided by the biomarker hypotheses developed here. We are currently enrolling patients for treatment with UCT-03-008 in a Phase 1 clinical trial of advanced solid tumors (NCT05103046). Citation Format: Neil A. O'Brien, Martina S. McDermott, Brendan M. O'Boyle, Justin A. Hilf, Oliver Loson, Kevin Chau, Weiping Jia, Naeimeh Kamranpour, Tong Luo, Raul Ayala, Shawnt Issakhanian, John A. Glaspy, Brian M. Stoltz, Dennis J. Slamon. Development and characterization of a novel CDK4/6 inhibitor for the treatment of CDK4/6 dependent cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5991.

Abstract 6346: Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy

Abstract OX40, tumor necrosis factor receptor superfamily member 4, is an immune co-stimulatory receptor, together with its ligand OX40L, forming OX40-OX40L trimer-trimer complex between the surface of T-cells (OX40) and APCs/NKs (OX40L). OX40 expresses constitutively on Treg as well as on conventional Teff, particularly within TME. OX40/OX40L interactions directly triggers conventional T cell activation via NF-kB1 pathway and inhibits bcl-x and survivin to prevent apoptosis; and it also downregulates FoxP3/CTLA4, thus Treg function. OX40 agonistic antibodies have also been shown to activate tumor immunity and increase anti-tumor activity in preclinical models. Here we report that we identified/constructed a novel OX40-agonistic humanized IgG1 monoclonal antibody (mAb), HX011, with high affinity through hybridoma technology. HX011 was subsequently confirmed of the similar binding properties and functions as the original mouse mAb clone, 9C12: 1) binding to human OX40 (EC50 ~0.012 nM; 2) similar function binding in the reporter assay (EC50 ~6.6 nM); but 3) has little blockade of the OX40L binding to OX40. In another word, HX011 is similar to BMS-986178 for the first two assays, but contrastingly different for the third assay. Non-competing with OX40L may actually maximize T-cell activation, a potential advantage over a competing antibody. In vivo pharmacology modeling of HX011 in the humanized syngeneic CRC model, MC38, where syngeneic C57BL/6 mouse mice were knocked in with huOX40 (“HuGEMM-OX40”), demonstrated strong anti-tumor activity (TGI ~100% at 1 mg/kg, twice weekly). HX011 is being further investigated as a potential new immunotherapy for the treatment of cancers. Citation Format: Hang Ke, Faming Zhang, Jialin Li, Feiyu Peng, Cen Chen, Henry Qixiang Li. Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6346.

Abstract 6158: BGB-24714, a novel oral IAP antagonist, displayed significant anti-tumor activities in preclinical models as a monotherapy and in combination with paclitaxel

Abstract Background: Evasion of apoptosis is identified as one of the essential hallmarks of cancer and upregulation of inhibitor of apoptosis proteins (IAPs) is one of the mechanisms by which tumor cells evade apoptosis. An oral SMAC mimetic and antagonist of cellular IAP1 (cIAP1) and X-linked IAP (XIAP), BGB-24714, is currently investigated in a phase 1a/1b oncology trial in patients with advanced or metastatic solid tumors (NCT05381909). Here, we evaluated the anti-tumor activity of BGB-24714 as a single agent or in combination with paclitaxel in preclinical models. Results: BGB-24714 effectively inhibited cIAP1 by inducing its degradation in MDA-MB-231 cells, with an EC50 of 2.5 nM. BGB-24714 also potently antagonized the inhibitory interaction of XIAP with caspase-9 and induced caspase-9 autoactivation in MDA-MB-231 cells, with an EC50 of 23 nM. In a total of 25 breast cancer cell lines treated with TNFα, BGB-24714 potently inhibited the in vitro proliferation of 5 breast cancer cells with EC50 < 100 nM. In pharmacodynamics studies, single dose administration of BGB-24714 significantly induced degradation of cIAP1 and antagonism of the XIAP: Smac interaction in the MDA-MB-231 xenograft model in a dose dependent manner. Using the same model, BGB-24714 exhibited dose-dependent anti-tumor activities as a single agent. The tumor growth inhibition rates were 30%, 52% and 73% in low to high dosage treatment groups. Furthermore, BGB-24714 at medium dosage level demonstrated synergized anti-tumor activity in HCC1806 xenograft model when used in combination with paclitaxel. In intermittent dosing study, BGB-24714 with the intermittent dosing schedule demonstrated significant but slightly less effective anti-tumor activity than the continuous dosing schedule. In summary, BGB-24714, as a novel oral IAP antagonist, showing significant anti-tumor activities in preclinical models, which is promising and warrants the testing of the compound in human. Citation Format: Jie Li, Nan Hu, Yilu Zhang, Xiaolong Yang, Minjuan Deng, Wenfeng Gong, Longbo Yin, Yong Liu, Yajuan Gao, Wei Wei, Xing Wang, Xinyi Liang, Yanwen Ma, Xuxing Sang, Chang Liu, Jingyuan Wang, Qianqian Lu, Fengtao Song, Xi Yuan, Yibing Wang, Jing Li, Wei Jin, Xuesong Liu, Xiaomin Song. BGB-24714, a novel oral IAP antagonist, displayed significant anti-tumor activities in preclinical models as a monotherapy and in combination with paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6158.

Abstract 6157: USP7 is a promising thersapeutic target for neuroblastoma

Abstract Background: The ubiquitin-proteasome system (UPS) plays an essential role in post-translational modification of proteins to maintain proteostasis. Ubiquitination of target protein is coordinated by different enzymes, which add and link ubiquitin to each other in specific ways that determine the fate of the protein for to be degraded or to be included into protein complex to induce signaling pathways. Ubiquitination is further regulated by deubiquitinases, which remove ubiquitin and rescue target protein from being degraded or diverted from it signaling role. UPS dysregulation has been linked to many human diseases, including cancer. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in immune response, tumor suppression and DNA repair. USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including in neuroblastoma (NB). Therefore, USP7 is a potential therapeutic target for NB. Some new specific USP7 inhibitors are highly potent and selective and have demonstrated significant antitumor activity in preclinical models of adult cancer. We hypothesize that inhibition of USP7 alone or in combination with epigenetic drugs like HDAC inhibitors will be more effective against NB tumor growth. To evaluate the efficacy of USP7 inhibition with specific USP7 inhibitors alone or with combination with epigenetic drugs like HDAC inhibitors (Saha) or chemotherapy against NB tumor growth, NB cell lines were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and cell viability using AlamarBlue assay. Apoptosis was measured by caspase cleavage using Caspase 3/7 apoptosis assay as well as by PARP cleavage by Western Blotting (WB). Changes in various proteins were measured by WB and co-immunoprecipitation were performed to analyze the ubiquitin status of specific protein targets regulated by USP7. Pharmacologic inhibition of USP7 resulted in significant decreases in both cell proliferation and cell viability in vitro only in p53 wild-type NB cell lines. There was no correlation seen between MYCN amplification status and treatment response. USP7 inhibition induced apoptosis as well as necroptosis with dose-dependent increase. Treatment with USP7 inhibitors led to decrease p53 ubiquitination leading increased of p53 protein expression in all sensitive cell lines tested and decreased MYCN protein level in MYCN-amplified cell lines. EZH2, which is a predominant target of USP7 is significantly downregulated with increasing dose of USP7 inhibitors. In addition, the combination of USP7 inhibitors with chemotherapy commonly used in NB or with combination with HDAC inhibitors showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, Karen Lee, Peter Zage. USP7 is a promising thersapeutic target for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6157.

Abstract 5176: CDDO-methyl ester redirects macrophage polarization and reduces lung tumor burden in a Nrf2-dependent manner

Abstract The cytoprotective Nrf2 pathway impacts immune cell function and has been proposed as a target for many inflammation-related diseases, but the effects of Nrf2 activation on immune cells in the cancer context are not well characterized. With Nrf2 activators under development and in clinical trials, it is critical to understand the influence of these drugs on cancer progression. While the anti-inflammatory nature of Nrf2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs and increase tumor cell survival. Up to 30% of human lung adenocarcinomas acquire mutations in the Nrf2 pathway which result in constitutive activation. However, triterpenoids, including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl), are potent pharmacological Nrf2 activators with demonstrated anti-cancer activity in preclinical models. Lung cancer is the leading cause of cancer-related mortality worldwide, and macrophages are the most common immune cell type in the lung tumor microenvironment. To investigate Nrf2 activation in macrophages in the context of lung cancer, bone marrow-derived macrophages (BMDMs) were isolated from wild type (WT) and Nrf2 knock out (KO) mice and cultured in conditioned media from lung cancer cells to produce a tumor-educated phenotype. The triterpenoid CDDO-Me had anti-inflammatory effects in BMDMs stimulated with the conventional cytokines IFN-γ and LPS. Conversely, CDDO-Me increased (p < 0.05) the M1 macrophage markers TNFα, IL-6, and MHC-II and decreased the M2 macrophage markers VEGF, CCL2, and CD206 in tumor-educated BMDMs in a Nrf2-dependent manner. The phenotypic changes were observed on transcription, protein, and surface marker levels. This context-dependent reversal of BMDM polarization suggests that Nrf2 activation has different outcomes in different environments. To test CDDO-Me in vivo, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 KO mice, and animals were fed either control diet or CDDO-Me (12.5-50 mg/kg of diet) for 16 weeks. CDDO-Me significantly (p < 0.05) decreased tumor number, size, and overall burden and reduced the histopathological severity of tumors in a Nrf2- and dose-dependent manner. Additionally, CDDO-Me increased the infiltration of CD64+ macrophages but decreased CD206+ expression in macrophages in the lungs of WT tumor-bearing mice. Interestingly, CD206 expression was higher on macrophages in the spleen of WT mice treated with CDDO-Me, suggesting a cancer context dependency. Future studies will evaluate the dependency of Nrf2 activation in macrophages for the anti-tumor activity of CDDO-Me. Citation Format: Jess Ann Moerland, Karen T. Liby. CDDO-methyl ester redirects macrophage polarization and reduces lung tumor burden in a Nrf2-dependent manner. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5176.