Induce Tumor Regression Research Articles

A CXCR4 partial agonist improves immunotherapy by targeting polymorphonuclear myeloid-derived suppressor cells and cancer-driven granulopoiesis.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that potently impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target1. Here, we fused a secreted CXCR4 partial agonist TFF2 to mouse serum albumin (MSA) and demonstrated that TFF2-MSA peptide synergized with anti-PD-1 to induce tumor regression or eradication, inhibited distant metastases, and prolonged survival in multiple gastric cancer (GC) models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track PMN-MDSC in vivo , we found TFF2-MSA selectively reduced the immunosuppressive Hdc-GFP + CXCR4 hi tumor PMN-MDSCs while preserving proinflammatory neutrophils, thereby boosting CD8 + T cell-mediated anti-tumor response together with anti-PD-1. Furthermore, TFF2-MSA systemically reduced PMN-MDSCs and bone marrow granulopoiesis. In contrast, CXCR4 antagonism plus anti-PD-1 failed to provide a similar therapeutic benefit. In GC patients, expanded PMN-MDSCs containing a prominent CXCR4 + LOX-1 + subset are inversely correlated with the TFF2 level and CD8 + T cells in circulation. Collectively, our studies introduce a strategy of using CXCR4 partial agonism to restore anti-PD-1 sensitivity in GC by targeting PMN-MDSCs and granulopoiesis.

Development of a First-in-Class DNMT1/HDAC Inhibitor with Improved Therapeutic Potential and Potentiated Antitumor Immunity.

Epigenetic therapies have emerged as a key paradigm for treating malignancies. In this study, a series of DNMT1/HDAC dual inhibitors were obtained by fusing the key pharmacophores from DNMT1 inhibitors (DNMT1i) and HDAC inhibitors (HDACi). Among them, compound (R)-23a demonstrated significant DNMT1 and HDAC inhibition both in vitro and in cells and largely phenocopied the synergistic effects of combined DNMT1i and HDACi in reactivating epigenetically silenced tumor suppressor genes (TSGs). This translated into a profound tumor growth inhibition (TGI = 98%) of (R)-23a in an MV-4-11 xenograft model, while displaying improved tolerability compared with single agent combination. Moreover, in a syngeneic MC38 mouse colorectal tumor model, (R)-23a outperformed the combinatory treatment in reshaping the tumor immune microenvironment and inducing tumor regression. Collectively, the novel DNMT1/HDAC dual inhibitor (R)-23a effectively reverses the cancer-specific epigenetic abnormalities and holds great potential for further development into cancer therapeutic agents.

ZG16 enhances the maturation of dendritic cells via induction of CD40 and contributes to the antitumor immunity in pancreatic cancer.

Dendritic cells (DCs) are critical mediators of antigen priming and T-cell activation. Zymogen granule protein 16 (ZG16) is demonstrated as an anti-oncogene in T-cell mediated antitumor immunity, but its effect on DCs is largely unknown. Herein, we wonder whether ZG16 affects the activation of DCs in pancreatic cancer. Firstly, the increased ZG16 expression was observed during the maturation of DCs derived from mouse bone marrow or human peripheral blood. Then, overexpression of ZG16 or exogenous introduction of recombinant ZG16 protein induced the expression of MHC II, CD86, CD84, and CCR7 on the surface of DCs, thereby facilitating the secretion of proinflammatory mediators IL-1β, IL-6, TNF-α, and IL-12/p70, supporting the promoting effect of ZG16 on DC maturation. By establishing the subcutaneous and orthotopic mouse models of pancreatic cancer, we confirmed that intraperitoneal injection of recombinant ZG16 protein (Re-mZG16) could induce tumor regression by stimulating DC maturation and enhancing antitumor responses of CD4 + , CD8 + , PD-1 + , and Ctla4+ cells. Besides, Re-mZG16 in combination with gemcitabine showed a synergistic effect in the treatment of pancreatic cancer. Mechanistically, we demonstrated that ZG16 inhibited the ubiquitination and degradation of CD40, which depended on the lectin domain of ZG16. In conclusion, this study provided a novel insight into the role of ZG16-CD40 axis in DC-based immunotherapy for pancreatic cancer.

Local administration of mRNA encoding cytokine cocktail confers potent anti-tumor immunity.

Immunotherapy using inflammatory cytokines, such as interleukin (IL)-2 and interferon (IFN)-α, has been clinically validated in treating various cancers. However, systemic immunocytokine-based therapies are limited by the short half-life of recombinant proteins and severe dose-limiting toxicities. In this study, we exploited local immunotherapy by intratumoral administration of lipid nanoparticle (LNP)-encapsulated mRNA cocktail encoding cytokines IL-12, IL-7, and IFN-α. The cytokine mRNA cocktail induced tumor regression in multiple syngeneic mouse models and anti-tumor immune memory in one syngeneic mouse model. Additionally, immune checkpoint blockade further enhanced the anti-tumor efficacy of the cytokine mRNAs. Furthermore, human cytokine mRNAs exhibited robust anti-tumor efficacy in humanized mouse tumor models. Mechanistically, cytokine mRNAs induced tumor microenvironment inflammation, characterized by robust T cell infiltration and significant inflammatory cytokine and chemokine production.

Intraperitoneal activation of myeloid cells clears ascites and reveals IL27-dependent regression of metastatic ovarian cancer.

Patients with metastatic ovarian cancer (OvCa) have a 5-year survival rate of less than 30% due to persisting dissemination of chemoresistant cells in the peritoneal fluid and the immunosuppressive microenvironment in the peritoneal cavity. Here, we report that intraperitoneal administration of β-glucan and IFNγ (BI) induced robust tumor regression in clinically relevant models of metastatic OvCa. BI induced tumor regression by controlling fluid tumor burden and activating localized antitumor immunity. β-glucan alone cleared ascites and eliminated fluid tumor cells by inducing intraperitoneal clotting in the fluid and Dectin-1-Syk-dependent NETosis in the omentum. In omentum tumors, BI expanded a novel subset of immunostimulatory IL27+ macrophages and neutralizing IL27 impaired BI efficacy in vivo. Moreover, BI directly induced IL27 secretion in macrophages where single agent treatment did not. Finally, BI extended mouse survival in a chemoresistant model and significantly improved chemotherapy response in a chemo-sensitive model. In summary, we propose a new therapeutic strategy for the treatment of metastatic OvCa.

Sequential intravenous and intracerebroventricular GD2-CAR T-cell therapy for H3K27M-mutated diffuse midline gliomas.

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the GD2 disialoganglioside and chimeric antigen receptor modified T-cells targeting GD2 (GD2-CART) eradicate DMGs in preclinical models. Arm A of the Phase I trial NCT04196413 administered one IV dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline glioma at two dose levels (DL1=1e6/kg; DL2=3e6/kg) following lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) GD2-CART infusions (10-30e6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability, and identification of a maximally tolerated dose of IV GD2-CART. Secondary objectives included preliminary assessments of benefit. Thirteen patients enrolled and 11 received IV GD2-CART on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CART manufacturing was successful for all patients. No dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric tumor reductions (52%, 54%, 91% and 100%). One patient exhibited a complete response ongoing for >30 months since enrollment. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. Sequential IV followed by ICV GD2-CART induced tumor regressions and neurological improvements in patients with DIPG and sDMG. DL1 was established as the maximally tolerated IV GD2-CART dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

Mechano-inhibition of endocytosis sensitizes cancer cells to Fas-induced Apoptosis

The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand, FasL. Although Fas is highly expressed in cancer cells, insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here, we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor, fasudil, that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL), fasudil promoted cancer cell apoptosis, but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis.

Venetoclax Induces BCL-2-Dependent Treg to TH17 Plasticity to Enhance the Antitumor Efficacy of Anti-PD-1 Checkpoint Blockade.

The specific BCL-2 small molecule inhibitor venetoclax induces apoptosis in a wide range of malignancies, which has led to rapid clinical expansion in its use alone and in combination with chemotherapy and immune-based therapies against a myriad of cancer types. While lymphocytes, and T cells in particular, rely heavily on BCL-2 for survival and function, the effects of small molecule blockade of the BCL-2 family on surviving immune cells is not fully understood. We aimed to better understand the effect of systemic treatment with venetoclax on regulatory T cells (Treg), which are relatively resistant to cell death induced by specific drugging of BCL-2 compared to other T cells. We found that BCL-2 blockade altered Treg transcriptional profiles and mediated Treg plasticity toward a TH17-like Treg phenotype, resulting in increased IL17A production in lymphoid organs and within the tumor microenvironment. Aligned with previously described augmented antitumor effects observed when combining venetoclax with anti-PD-1 checkpoint inhibition, we also demonstrated that Treg-specific genetic BCL-2 knockout combined with anti-PD-1 induced tumor regression and conferred overlapping genetic changes with venetoclax-treated Tregs. As long-term combination therapies using venetoclax gain more traction in the clinic, an improved understanding of the immune-modulatory effects caused by venetoclax may allow expansion of its use against malignancies and immune-related diseases.

MDB-79. THE DEUBIQUITINATING ENZYME USP7 COOPERATES WITH SONIC HEDGEHOG SIGNALING TO PROMOTE MEDULLOBLASTOMA

Abstract BACKGROUND Constitutive activation of the Sonic Hedgehog (SHH) signaling pathway during cerebellar development may lead to medulloblastoma (MB), one of the most common malignant pediatric brain tumor. Clinically, SHH-MB with mutations downstream of SMO or TP53-mutated are a real challenge and alternative targeted therapies are sorely needed for this group of patients. In SHH-MB, the transcription factor Atoh1 is constitutively overexpressed and essential for tumor initiation and progression. Previous work demonstrated that proteasomal-mediated degradation of Atoh1 induces tumor regression in vitro and in vivo, therefore deciphering mechanisms controlling Atoh1 stabilization should provide new avenues for drug development in SHH-MB. METHODS Using a proteomic approach, we identified the deubiquitinating enzyme Usp7 as a positive regulator of Atoh1 in MB cells. We then applied a SILAC-based quantitative ubiquitinomics to investigate the Usp7 target landscape in SHH-MB. We also employed several biochemical approaches to demonstrate the functional interaction between Atoh1 and Usp7 in SHH-MB. In parallel, we validated the impact of Usp7 depletion in vitro and in vivo using relevant SHH-MB models. Last, we investigated large transcriptomic and proteomic datasets to unveil the prognostic value of Usp7 in SHH-MB human tumors. RESULTS We demonstrate that Usp7 binds and accumulates Atoh1 protein by counteracting its ubiquitylation. Post-natal electroporation in vivo directly into the developing cerebella also indicates that overexpression of Usp7 promotes proliferation, whereas in the absence of Atoh1 Usp7 was no longer able to spread cell growth, demonstrating a functional link between the Usp7-Atoh1 axis. In vivo, disruption of Usp7 expression in SHH-MB blocks tumor proliferation. Pharmacological inhibition of Usp7 triggers tumor growth in a syngeneic SHH MB mouse model. Lastly, SHH-MB cells with either TP53 or SMO downstream mutations are sensitive to Usp7 inhibition. CONCLUSIONS Overall, our data pinpoint Usp7 as a promising target against Atoh1, a master regulator of SHH-MB.

DIPG-47. SEQUENTIAL INTRAVENOUS AND INTRACEREBROVENTRICULAR GD2-CAR T-CELL THERAPY FOR H3K27M-MUTATED DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND H3K27M-mutant diffuse midline gliomas (DMGs) express uniformly high levels of the GD2 disialoganglioside. In preclinical models, chimeric antigen receptor modified T-cells (CAR T-cells) targeting GD2 robustly regressed orthotopically xenografted DMGs. METHODS This Phase I trial (NCT04196413) administered one IV dose of autologous T-cells transduced with a GD2-CAR retroviral vector to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline gliomas at two dose levels (DL1=1e6 GD2-CAR T-cells/kg; DL2=3e6 GD2-CAR T-cells/kg) following standard lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit following IV infusion were eligible for subsequent intracerebroventricular (ICV) GD2-CAR T-cell infusions (10-30e6 GD2-CAR T-cells). Primary objectives were to determine feasibility of manufacturing, assess tolerability of IV GD2-CAR T-cells in patients with DIPG and sDMG, and identify a maximally tolerated dose of IV GD2-CAR T-cells following lymphodepleting chemotherapy. Secondary objectives included preliminary assessments of benefit. Here we report the final results of Arm A. RESULTS Thirteen patients enrolled and 11 received one IV GD2-CAR T infusion on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CAR T-cells were successfully manufactured for each patient. After IV infusion, no dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric reductions of 52%, 54%, 91% and 100%. One patient exhibited a complete response durable for >30 months since therapy began. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. CONCLUSIONS Sequential IV followed by ICV GD2-CAR T-cells infusions induced tumor regressions and neurological improvements. DL1 was established as the maximally tolerated IV GD2-CAR T-cell dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

Receptor tyrosine kinase inhibition leads to regression of acral melanoma by targeting the tumor microenvironment.

Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is biologically distinct, has an equal incidence across genetic ancestries, typically presents in advanced stage disease, is less responsive to therapy, and has an overall worse prognosis. Independent analysis of published genomic and transcriptomic sequencing identified that receptor tyrosine kinase (RTK) ligands and adapter proteins are frequently amplified, translocated, and/or overexpressed in AM. To target these unique genetic changes, a zebrafish acral melanoma model was exposed to a panel of narrow and broad spectrum multi-RTK inhibitors, revealing that dual FGFR/VEGFR inhibitors decrease acral-analogous melanocyte proliferation and migration. The potent pan-FGFR/VEGFR inhibitor, Lenvatinib, uniformly induces tumor regression in AM patient-derived xenograft (PDX) tumors but only slows tumor growth in CM models. Unlike other multi-RTK inhibitors, Lenvatinib is not directly cytotoxic to dissociated AM PDX tumor cells and instead disrupts tumor architecture and vascular networks. Considering the great difficulty in establishing AM cell culture lines, these findings suggest that AM may be more sensitive to microenvironment perturbations than CM. In conclusion, dual FGFR/VEGFR inhibition may be a viable therapeutic strategy that targets the unique biology of AM.

Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Pharmacodynamics of venetoclax plus cirtuvivint a first-in-class splice variant regulator.

e15093 Background: NSC835563 (SM08502, cirtuvivint) is a first-in-class pan CDC-like kinase (CLK) and dual specificity tyrosine kinase (DYRK) inhibitor that targets alternative splicing of genes in multiple pathways and modifies their protein expression. Preclinical screening of hematologic malignancy models has shown sensitivity to cirtuvivint, leading to proposed clinical trials in AML in combinations with venetoclax. We performed pharmacodynamic studies to identify probable mechanisms of combination activity in AML models. Methods: In vivo tolerability, efficacy and pharmacodynamic (PD) studies were performed in two AML xenograft models, MV411 and KG1a in NSG mice, at 6.25-25 mg/kg PO cirtuvivint and 25 mg/kg PO venetoclax. Body weights (BW) and tumor volumes were recorded periodically to assess toxicity and anti-tumor efficacy. Tumor tissue samples were collected at 6 and 24 h post venetoclax to assess PD response. Multiplexed analysis of apoptosis pathway proteins/protein-protein complexes was performed by clinically validated assays (PMID 26446940). Results: Cirtuvivint alone at high doses (25 mg/kg) caused tumor regressions in MV411 but only a modest tumor growth inhibition (TGI) in KG1a, a resistant AM model. Importantly, the lowest dose of 6.25 mg/kg cirtuvivint combined with 25 mg/kg venetoclax led to tumor regression in the MV411 model and a moderate TGI in KG1a. Cirtuvivint alone caused a transient 25% reduction (p<0.05) in Mcl-1 levels at 6 h post dose in MV411. The venetoclax combination triggered a sustained (for 24 h) 50-75% disruption of Bcl-2 and Mcl-1 complexes with Bax and Bim, resulting in >20-fold increased mitochondrial Bax/Bak heterodimer and >10-fold increased cytosolic cleaved caspase-3. In KG1a, combination treatment resulted in only modest (<20%) changes in biomarkers. Overall, biomarkers provided evidence of the combination MoA. Conclusions: Venetoclax and cirtuvivint in combination effectively induced tumor regressions at low tolerable doses in an AML model. Mitochondrial Bax/Bak heterodimer provided a sensitive biomarker of combination activity. Cirtuvivint contributed its combinatorial effect primarily through disruption of Bim complexes and inhibition of Mcl-1. PD biomarkers identified in this study can confirm MoA of drug combination in ongoing clinical trials.

Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1.

Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.

CHES1 modulated tumorigenesis and senescence of pancreas cancer cells through repressing AKR1B10

Pancreatic ductal adenocarcinoma (PDAC), is characteristic by a heterogeneous tumor microenvironment and gene mutations, conveys a dismal prognosis and low response to chemotherapy and immunotherapy. Here, we found that checkpoint suppressor 1 (CHES1) served as a tumor repressor in PDAC and was associated with patient prognosis. Functional experiments indicated that CHES1 suppressed the proliferation and invasion of PDAC by modulating cellular senescence. To further identify the downstream factor of CHES1 in PDAC, label-free quantitative proteomics analysis was conducted, which showed that the oncogenic Aldo-keto reductase 1B10 (AKR1B10) was transcriptionally repressed by CHES1 in PDAC. And AKR1B10 facilitated the malignant activity and repressed senescent phenotype of PDAC cells. Moreover, pharmaceutical inhibition of AKR1B10 with Oleanolic acid (OA) significantly induced tumor regression and sensitized PDAC cells to gemcitabine, and this combined therapy did not cause obvious side effects. Rescued experiments revealed that CHES1 regulated the tumorigenesis and gemcitabine sensitivity through AKR1B10-mediated senescence in PDAC. In summary, this study revealed that the CHES1/AKR1B10 axis modulated the progression and cellular senescence in PDAC, which might provide revenues for drug-targeting and senescence-inducing therapies for PDAC.

Abstract PO1-04-14: PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts

Abstract Paclitaxel is a first-line chemotherapy for hormone receptor positive (HR+) metastatic breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Response rate to paclitaxel ranges between 20-40%, and most patients progress due to intrinsic or acquired resistance, leaving them with limited treatment choices. Therapeutic strategies to overcome paclitaxel resistance and extend its clinical benefit are urgently needed. Paclitaxel-resistant tumors have increased expression of polo-like kinase 1 (PLK1), a serine-threonine-protein kinase that regulates mitosis and cell cycle progression. PLK1 has also been shown to mediate resistance to CDK4/6 inhibitor palbociclib in HR+ breast cancer. Onvansertib is an orally bioavailable, highly potent, and selective PLK1 inhibitor in clinical development. Onvansertib exhibited synergy in vitro and showed potent anti-tumor activity in vivo in combination with paclitaxel in ovarian cancer and triple negative breast cancer (TNBC) preclinical models. A phase 1b/2 clinical trial is ongoing to evaluate the safety and efficacy of onvansertib plus paclitaxel in advanced TNBC (NCT05383196). Here we investigated whether the potential of onvansertib and paclitaxel combination could be extended to HR+ breast cancer. Co-treatment with onvansertib and paclitaxel synergistically inhibited the viability of HR+ breast cancer cell lines (MCF-7, T-47D, EFM-19, CAMA-1, ZR-75-1). Compared to the single agents, the combination induced increased apoptosis. The combination was further tested in vivo in patient-derived xenograft (PDX) models. To this end, six HR+ breast cancer PDXs with intrinsic or acquired resistance to palbociclib were established from primary breast tumors (n=1) or metastatic bone biopsies (n=5). The PDX tumors were engrafted subcutaneously in nude mice and the effect of onvansertib (45 mg/kg, oral, 5 days a week) and paclitaxel (15-25mg/kg, IP, once a week) was investigated as monotherapy and in combination. The combination of onvansertib and paclitaxel was tolerated with minimal toxicity and showed enhanced anti-tumor activity compared to either agent alone in all 6 PDX models. Paclitaxel and onvansertib monotherapies had little to no anti-tumor activity in 4 out of 6 PDX models (HBCx124palboR25, HBCx-139 palboR5, HBCx-202 and HBCx-131). Importantly, the combination of onvansertib and paclitaxel induced strong anti-tumor activity in these 4 models. Tumor regression was observed in the 2 PDX models with acquired resistance to palbociclib, HBCx-124palboR25 and HBCx-139palboR5, with 62% (5/8 mice) and 54% (6/11 mice) complete response rates, respectively. The combination showed tumor stasis in HBCx-131 and induced tumor regression in HBCx-202, established from patient resistant to palbociclib. In 2 of the 6 PDXs, paclitaxel exhibited anti-tumor activity, inducing tumor regression in HBCx-137paboR26 and tumor stasis in HBCx-86. In comparison, the combination of onvansertib and paclitaxel had greater activity, inducing tumor regression in both models with a higher rate of complete response than the monotherapy. Complete response rates for paclitaxel versus the combination were 62% and 100% in the HBCx-137palboR26 model and 0% and 67% in the HBCx-86 model. Notably, the antitumor activity of the onvansertib and paclitaxel combination was very durable, showing a robust delay in tumor relapse after stopping therapy. Together, our data strongly support that combining paclitaxel with the PLK1 inhibitor onvansertib extends its benefit and overcomes paclitaxel resistance, and represents a promising therapeutic strategy for HR+ breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Citation Format: Maya Ridinger, Pierre Painsec, Sreeja Sreekumar, Dalia Gonzalez, Davis Klein, Laura Sourd, Elodie Montaudon, Paul Cottu, Tod Smeal, Elisabetta Marangoni. PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts [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 PO1-04-14.

Abstract PO5-19-07: A Single Arm Phase 2 Study of Peri-Operative Immune Checkpoint Inhibition and Cryoablation in Women With Hormone Receptor-Negative, HER2-Negative Early Stage/Resectable Breast Cancer

Abstract Background: Triple negative breast cancer (TNBC) is a biologically distinct subtype with a high risk of early relapse. Patients not achieving a pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) have a 3-year event free survival (EFS) of < 60%. Approximately 1/3 of patients receiving NAC with pembrolizumab-mediated immune therapy will not achieve a pCR and of those, approximately 1/3 will experience a recurrence within 3 years. Physical disruption of tumors with local strategies such as cryoablation (cryo) induces inflammation and releases antigens that can activate tumor-specific immune responses. The combination of cryo with immune checkpoint inhibition (ICI) has been shown to induce tumor regression and prevented tumor re-challenge in pre-clinical models. Clinical studies have also demonstrated that the combination of pre-operative cryo with ICI is safe in women with operable, early-stage breast cancer and generates intra-tumoral and systemic immune responses (NCT01502592, NCT02833233). This multi-center, single arm, phase 2 study is currently evaluating the impact of cryo with standard-of-care pembrolizumab (pembro) in women with residual TNBC after taxane-based NAC, a group at high risk of early relapse (NCT03546686). Methods: Eligible women are ≥18y, with ER < 10%, PR < 10%, HER2 negative (per ASCO/CAP definition), ≥ 1.0 cm, residual operable disease after taxane-based NAC. Pts undergo percutaneous, image-guided cryo with concurrent research core biopsy 7-10 days prior to surgery and receive peri-operative pembro per standard-of-care. Adjuvant capecitabine or olaparib is recommended for all patients per local standard-of-care. Patients are stratified by NAC platinum administration, NAC anthracycline administration, and clinical nodal status (positive versus negative). The primary endpoint is 3-year Event Free Survival (EFS). Secondary endpoints include Invasive Disease-Free Survival (IDFS), Distant Disease-Free Survival (DDFS), overall survival (OS) and safety. Exploratory correlative studies will be performed on tumor and serum to characterize the immunologic impact of the intervention and to explore predictors of efficacy and toxicity. Funding sources: Conquer Cancer Foundation, Breast Cancer Research Foundation, Boston Scientific Citation Format: Heather McArthur, Elizabeth Comen, Yolanda Bryce, Stephen Solomon, Sangeetha Reddy, Isaac Chan, Baṣak Dogan, Dawn Klemow, Nisha Unni, Jorge Henrique Santos Leal, Cristal Martinez, Reva Basho, Dorothy Park, Philomena McAndrew, Brigid Larkin, David Page, William Mills, Staci Mellinger, Nicole Fredrich, Nicole Moxon, Lynette Currie, Meredith Carter, Melissa Ramos, Stephanie Rice, Sujata Patil, Margaret Gatti-Mays, Larry Norton. A Single Arm Phase 2 Study of Peri-Operative Immune Checkpoint Inhibition and Cryoablation in Women With Hormone Receptor-Negative, HER2-Negative Early Stage/Resectable Breast Cancer [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 PO5-19-07.

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Abstract LB067: Tumor-reactive CD8 TIL with an exhausted phenotype can be expanded and regress human tumors

Abstract Tumor-reactive human CD8 T cells are found predominantly in the tumor microenvironment with an exhausted phenotype (extremely high levels of CD39, PD-1, CTLA-4, and TIM-3). The tumor-reactive cells can be enriched from patients’ tumors by sorting CD8+ TIL that express both CD39 and CD103. Our unique expansion method allowed these phenotypically exhausted cells to grow from thousands into billions. After expansion the CD39 and CD103 Double Positive (DP) CD8 TIL can recognize/kill autologous tumor cells in vitro. We tested whether the CD8 DP TIL could recognize/kill autologous tumor in vivo using a xenograft model with immune-compromised mice that constitutively secrete human IL-2 (NOG-hIL-2). The CD8 DP TIL were able to persist long-term, traffic to the tumor site, recognize autologous tumor, and facilitate tumor regression. Human IL-2 was necessary for their long-term survival in the periphery and for tumor regression. These preclinical data were the basis for a human clinical trial design for the adoptive transfer of CD8 DP TIL (product termed, AGX148). We recently opened a Phase 1 clinical trial using the CD39/103 DP CD8 TIL in a first-in-human protocol for adults with solid tumors (NCT05902520). The trial design has three cohorts based on giving IL-2 for 2 weeks, 3 weeks, or 4 weeks after adoptive transfer with six patients/cohort (3 receiving DP TIL alone and 3 receiving DP TIL combined with PD-1 siRNA knockdown with the INTASYLTM compound PH-762). The first 3 patients have been treated and received 27, 40, and 8 billion DP TIL, respectively. All 3 patients had failed standard therapy prior to their enrollment, including checkpoint blockade. No serious adverse events were observed in the first three patients. Two of three patients had partial responses 29 days after treatment and the third patient had stable disease for >56 days. Blood draws and tumor biopsies were obtained prior to and after treatment and we are currently assessing the CD8 T cell repertoire (TCRseq) and phenotype from these patient samples. In conclusion, this is the first study showing that CD8 T cells with an exhausted phenotype isolated from human tumors can be expanded to billions and induce tumor regression in vivo. Citation Format: Andrew Weinberg, Brendan Curti, Ryan Montler, Colin Thalhofer, Elaine Ballinger, Jessica Cross, Eric Tran, Nelson Sanjuan, Matthew Taylor, Christopher Fountain, Roxanne Payne, James Cardia, Nicholas Morris. Tumor-reactive CD8 TIL with an exhausted phenotype can be expanded and regress human tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB067.

Abstract LB213: NF1 loss promotes bone metastasis in estrogen receptor positive breast cancer

Abstract Up to 80% of breast cancer (BCa) expresses estrogen receptor-α (ER). Over 60% ER+ BCa metastasizes to the bone and are challenging to treat. This study investigates what drives bone metastasis by focusing on a key tumor suppressor neurofibromin (NF1), a GTPase-activating protein that represses RAS signaling. Earlier, we reported that NF1 acts as a transcriptional corepressor for ER in BCa. Our group established that combination fulvestrant (selective ER degrader) and binimetinib (MEK-inhibitor) induces tumor regression in NF1lowER+ models. This combination is now in clinical trial, NCT05554354 (NCI ComboMATCH). Numerous studies indicate that the NF1 loss may impact bone health by altering the equilibrium between bone formation and resorption. Since, bone is the primary site of metastasis in 50-70% of ER+ BCa, understanding the role of NF1 loss in bone metastases becomes imperative. An osteolytic vicious cycle may drive bone metastasis, where cancer cells in the bone stimulate the bone-destructing osteoclasts (OC); leading to subsequent bone deterioration, which in turn, releases growth factors to further promote tumor growth. OCs and many cells in the bone are ER+. We thus hypothesize that deregulated RAS and ER signaling by NF1 loss can drive ER+ bone metastasis by promoting an osteolytic vicious cycle. To assess the clinical impact of NF1 loss on bone metastasis, we analyzed metastatic BCa patient data from cBioPortal and the MSK-EMC cohort. To define how NF1 loss affect bone metastasis, NF1 knockout (KO) ER+ BCa cells were tested in vitro and in vivo for bone tropism, growth in bone and effect on the vicious cycle. Analyses of clinical data suggest that NF1 copy number (CN) loss in ER+ tumors associates with accelerated metastasis to the bone. Additionally, NF1 CN loss and mutations are more prevalent in bone metastasis BCa patients. Lower levels of NF1 mRNA are correlated with shorter duration of bone metastasis-free survival.Our preclinical studies further supported the driver role of NF1 in bone metastasis. NF1KO ER+ BCa cells migrate more efficiently toward the bone in vitro compared to their NF1+ counterparts. When injected into the mammary fat pads of mice, NF1KO cells more frequently metastasized to the bone compared to NF1+ cells. When NF1KO cells were delivered to the mouse femur by intra-iliac injection, they grew more efficiently and induced more bone degradation. This is consistent with our in vitro results, which show that NF1KO ER+ BCa cells can more efficiently promote OC formation from progenitor cells. For this experiment, Mass-spectrometry based unbiased profiling of the conditioned medium showed that osteolytic factors, such as ICAM1 and OSTF1, were enriched in the NF1KO medium. Hence, we conclude that the loss of NF1 deregulates RAS and ER, leading to increased osteolytic factors, that drive bone metastasis. Co-targeting RAS and ER can be a potential treatment for NF1 loss-induced bone metastasis in ER+ BCa. Citation Format: Zifan Zhao, Ze-Yi Zheng, Jonathan Lei, Matthew J. Baik, Yi-Hsuan Wu, Junkai Wang, Igor Bado, Meenakshi Anurag, Bing Zhang, Xiang Zhang, Eric Chang. NF1 loss promotes bone metastasis in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB213.