Anna E. Vilgelm

Combinatorial approach to cancer immunotherapy: strength in numbers

Immune‐checkpoint blockade therapy with antibodies targeting CTLA‐4 and PD‐1 has revolutionized melanoma treatment by eliciting responses that can be remarkably durable and is now advancing to other malignancies. However, not all patients respond to immune‐checkpoint inhibitors. Extensive preclinical evidence suggests that combining immune‐checkpoint inhibitors with other anti‐cancer treatments can greatly improve the therapeutic benefit. The first clinical success of the combinatorial approach to cancer immunotherapy was demonstrated using a dual‐checkpoint blockade with CTLA‐4 and PD‐1 inhibitors, which resulted in accelerated FDA approval of this therapeutic regimen. In this review, we discuss the combinations of current and emerging immunotherapeutic agents in clinical and preclinical development and summarize the insights into potential mechanisms of synergistic anti‐tumor activity gained from animal studies. These promising combinatorial partners for the immune‐checkpoint blockade include therapeutics targeting additional inhibitory receptors of T cells, such as TIM‐3, LAG‐3, TIGIT, and BTLA, and agonists of T cell costimulatory receptors 4‐1BB, OX40, and GITR, as well as agents that promote cancer cell recognition by the immune system, such as tumor vaccines, IDO inhibitors, and agonists of the CD40 receptor of APCs. We also review the therapeutic potential of regimens combining the immune‐checkpoint blockade with therapeutic interventions that have been shown to enhance immunogenicity of cancer cells, including oncolytic viruses, RT, epigenetic therapy, and senescence‐inducing therapy.

Preparation of (-)-Nutlin-3 using enantioselective organocatalysis at decagram scale.

Chiral nonracemic cis-4,5-bis(aryl)imidazolines have emerged as a powerful platform for the development of cancer chemotherapeutics, stimulated by the Hoffmann-La Roche discovery that Nutlin-3 can restore apoptosis in cells with wild-type p53. The lack of efficient methods for the enantioselective synthesis of cis-imidazolines, however, has limited their more general use. Our disclosure of the first enantioselective synthesis of (-)-Nutlin-3 provided a basis to prepare larger amounts of this tool used widely in cancer biology. Key to the decagram-scale synthesis described here was the discovery of a novel bis(amidine) organocatalyst that provides high enantioselectivity at warmer reaction temperature (-20 °C) and low catalyst loadings. Further refinements to the procedure led to the synthesis of (-)-Nutlin-3 in a 17 g batch and elimination of all but three chromatographic purifications.

Mdm2 and Aurora Kinase A Inhibitors Synergize to Block Melanoma Growth by Driving Apoptosis and Immune Clearance of Tumor Cells

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

Connecting the Dots: Therapy-Induced Senescence and a Tumor-Suppressive Immune Microenvironment

BACKGROUND Tumor cell senescence is a common outcome of anticancer therapy. Here we investigated how therapy-induced senescence (TIS) affects tumor-infiltrating leukocytes (TILs) and the efficacy of immunotherapy in melanoma. METHODS Tumor senescence was induced by AURKA or CDK4/6 inhibitors (AURKAi, CDK4/6i). Transcriptomes of six mouse tumors with differential response to AURKAi were analyzed by RNA sequencing, and TILs were characterized by flow cytometry. Chemokine RNA and protein expression were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Therapeutic response was queried in immunodeficient mice, in mice with CCL5-deficient tumors, and in mice cotreated with CD137 agonist to activate TILs. CCL5 expression in reference to TIS and markers of TILs was studied in human melanoma tumors using patient-derived xenografts (n = 3 patients, n = 3 mice each), in AURKAi clinical trial samples (n = 3 patients, before/after therapy), and in The Cancer Genome Atlas (n = 278). All statistical tests were two-sided. RESULTS AURKAi response was associated with induction of the immune transcriptome (P = 3.5 x 10-29) while resistance inversely correlated with TIL numbers (Spearman r = -0.87, P < .001). AURKAi and CDK4/6i promoted the recruitment of TILs by inducing CCL5 secretion in melanoma cells (P ≤ .005) in an NF-κB-dependent manner. Therapeutic response to AURKAi was impaired in immunodeficient compared with immunocompetent mice (0% vs 67% tumors regressed, P = .01) and in mice bearing CCL5-deficient vs control tumors (P = .61 vs P = .02); however, AURKAi response was greatly enhanced in mice also receiving T-cell-activating immunotherapy (P < .001). In human tumors, CCL5 expression was also induced by AURKAi (P ≤ .02) and CDK4/6i (P = .01) and was associated with increased immune marker expression (P = 1.40 x 10-93). CONCLUSIONS Senescent melanoma cells secret CCL5, which promotes recruitment of TILs. Combining TIS with immunotherapy that enhances tumor cell killing by TILs is a promising novel approach to improve melanoma outcomes.

Combining an Aurora Kinase Inhibitor and a Death Receptor Ligand/Agonist Antibody Triggers Apoptosis in Melanoma Cells and Prevents Tumor Growth in Preclinical Mouse Models

Purpose: Preclinical studies show that inhibition of aurora kinases in melanoma tumors induces senescence and reduces tumor growth, but does not cause tumor regression. Additional preclinical models are needed to identify agents that will synergize with aurora kinase inhibitors to induce tumor regression. Experimental Design: We combined treatment with an aurora kinase A inhibitor, MLN8237, with agents that activate death receptors (Apo2L/TRAIL or death receptor 5 agonists) and monitored the ability of this treatment to induce tumor apoptosis and melanoma tumor regression using human cell lines and patient-derived xenograft (PDX) mouse models. Results: We found that this combined treatment led to apoptosis and markedly reduced cell viability. Mechanistic analysis showed that the induction of tumor cell senescence in response to the AURKA inhibitor resulted in a decreased display of Apo2L/TRAIL decoy receptors and increased display of one Apo2L/TRAIL receptor (death receptor 5), resulting in enhanced response to death receptor ligand/agonists. When death receptors were activated in senescent tumor cells, both intrinsic and extrinsic apoptotic pathways were induced independent of BRAF, NRAS, or p53 mutation status. Senescent tumor cells exhibited BID-mediated mitochondrial depolarization in response to Apo2L/TRAIL treatment. In addition, senescent tumor cells had a lower apoptotic threshold due to decreased XIAP and survivin expression. Melanoma tumor xenografts of one human cell line and one PDX displayed total blockage of tumor growth when treated with MLN8237 combined with DR5 agonist antibody. Conclusions: These findings provide a strong rationale for combining senescence-inducing therapeutics with death receptor agonists for improved cancer treatment. Clin Cancer Res; 21(23); 5338–48. ©2015 AACR.

PI3K Inhibition Reduces Mammary Tumor Growth and Facilitates Antitumor Immunity and Anti-PD1 Responses

Purpose: Metastatic breast cancers continue to elude current therapeutic strategies, including those utilizing PI3K inhibitors. Given the prominent role of PI3Kα,β in tumor growth and PI3Kγ,δ in immune cell function, we sought to determine whether PI3K inhibition altered antitumor immunity. Experimental Design: The effect of PI3K inhibition on tumor growth, metastasis, and antitumor immune response was characterized in mouse models utilizing orthotopic implants of 4T1 or PyMT mammary tumors into syngeneic or PI3Kγ-null mice, and patient-derived breast cancer xenografts in humanized mice. Tumor-infiltrating leukocytes were characterized by IHC and FACS analysis in BKM120 (30 mg/kg, every day) or vehicle-treated mice and PI3Kγnull versus PI3KγWT mice. On the basis of the finding that PI3K inhibition resulted in a more inflammatory tumor leukocyte infiltrate, the therapeutic efficacy of BKM120 (30 mg/kg, every day) and anti-PD1 (100 μg, twice weekly) was evaluated in PyMT tumor–bearing mice. Results: Our findings show that PI3K activity facilitates tumor growth and surprisingly restrains tumor immune surveillance. These activities could be partially suppressed by BKM120 or by genetic deletion of PI3Kγ in the host. The antitumor effect of PI3Kγ loss in host, but not tumor, was partially reversed by CD8+ T-cell depletion. Treatment with therapeutic doses of both BKM120 and antibody to PD-1 resulted in consistent inhibition of tumor growth compared with either agent alone. Conclusions: PI3K inhibition slows tumor growth, enhances antitumor immunity, and heightens susceptibility to immune checkpoint inhibitors. We propose that combining PI3K inhibition with anti-PD1 may be a viable therapeutic approach for triple-negative breast cancer. Clin Cancer Res; 23(13); 3371–84. ©2016 AACR.

Combined therapies that induce senescence and stabilize p53 block melanoma growth and prompt antitumor immune responses

We recently demonstrated that dual therapy combining AURKA and MDM2 antagonists is effective against melanoma in preclinical settings. Notably, besides inducing apoptosis, this regimen led to tumor senescence and stimulated the hosts antitumor immune defenses. Treatments leveraging both cancer cell-intrinsic and extrinsic antitumor mechanisms can improve melanoma therapeutic outcomes.

Loss of CXCR4 in Myeloid Cells Enhances Antitumor Immunity and Reduces Melanoma Growth through NK Cell and FASL Mechanisms

NK-cell antitumor activity is enhanced by myeloid-CXCR4 deletion, revealing a pathway by which this receptor may contribute to tumor surveillance suppression and promotion of metastasis. This pathway provides a rationale for the clinical application of CXCR4 antagonists. The chemokine receptor, CXCR4, is involved in cancer growth, invasion, and metastasis. Several promising CXCR4 antagonists have been shown to halt tumor metastasis in preclinical studies, and clinical trials evaluating the effectiveness of these agents in patients with cancer are ongoing. However, the impact of targeting CXCR4 specifically on immune cells is not clear. Here, we demonstrate that genetic deletion of CXCR4 in myeloid cells (CXCR4MyeΔ/Δ) enhances the antitumor immune response, resulting in significantly reduced melanoma tumor growth. Moreover, CXCR4MyeΔ/Δ mice exhibited slowed tumor progression compared with CXCR4WT mice in an inducible melanocyte BrafV600E/Pten−/− mouse model. The percentage of Fas ligand (FasL)–expressing myeloid cells was reduced in CXCR4MyeΔ/Δ mice as compared with myeloid cells from CXCR4WT mice. In contrast, there was an increased percentage of natural killer (NK) cells expressing FasL in tumors growing in CXCR4MyeΔ/Δ mice. NK cells from CXCR4MyeΔ/Δ mice also exhibited increased tumor cell killing capacity in vivo, based on clearance of NK-sensitive Yac-1 cells. NK cell–mediated killing of Yac-1 cells occurred in a FasL-dependent manner, which was partially dependent upon the presence of CXCR4MyeΔ/Δ neutrophils. Furthermore, enhanced NK cell activity in CXCR4MyeΔ/Δ mice was also associated with increased production of IL18 by specific leukocyte subpopulations. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell–mediated tumor surveillance and thereby enhance tumor growth. Systemic delivery of a peptide antagonist of CXCR4 to tumor-bearing CXCR4WT mice resulted in enhanced NK-cell activation and reduced tumor growth, supporting potential clinical implications for CXCR4 antagonism in some cancers. Cancer Immunol Res; 6(10); 1186–98. ©2018 AACR.

MDM2 Antagonists Counteract Drug-Induced DNA Damage

Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.

Abstract 513: The link between polyploidy and replication stress in melanoma

Genomic instability is a hallmark of cancer implicated in tumor evolution and resistance to therapy. However, the molecular mechanisms that underlie genomic instability are still poorly understood. One common feature of cancer cells is an elevated genomic content (polyploidy) which is also associated with the acquisition of therapy resistance. Here we demonstrate a link between genomic instability and polyploidization in melanoma cells. We show that polyploidy induced by treatment with small molecule inhibitors of various mitotic kinases, such as AURKA, AURKB and PLK1, leads to DNA damage in cells with elevated (>4n) DNA content. This DNA damage results from replication stress exacerbated by limited activation of p53 in malignant melanocytes. Pharmacological induction of p53 in polyploid cells using an MDM2 antagonist reduced DNA damage by blocking re-replication of the polyploid genome as a result of p53-mediated p21 activation and transactivation of DNA repair genes. Notably we found that p21 blockade using siRNA knockdown or genetic knockout shifted polypoid cell response to p53 activation from cytostatic to cytotoxic. As a result, p21-deficient cells exhibited enhanced sensitivity to mitotic blockade combined with p53 induction. Furthermore, TCGA dataset analysis showed poor progression free survival in melanoma patients with high p21 protein expression. These data argue for administering mitotic inhibitors and MDM2 antagonists, which are currently in clinical development, in conjunction with agents that target p21. In summary, our data here reveal that polyploidization can be a mechanism for induction of DNA damage and genomic instability associated with drug resistance in cancer and suggest a novel strategy for targeting these pathways to improve melanoma therapy. Citation Format: Anna E. Vilgelm, C. Andrew Johnson, Kiran Malikayil, Dayanidhi Raman, David Flaherty, Brian Higgins, Ann Richmond. The link between polyploidy and replication stress in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 513. doi:10.1158/1538-7445.AM2017-513