Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy.

BACKGROUND: The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice. METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated. RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes. CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.

Nanoparticle-enhanced proton beam immunoradiotherapy drives immune activation and durable tumor rejection.

The combination of radiation therapy (RT) and immunotherapy has emerged as a promising treatment option in oncology. Historically, x-ray radiation (XRT) has been the most commonly used form of RT. However, proton beam therapy (PBT) is gaining recognition as a viable alternative, as it has been shown to produce similar outcomes to XRT while minimizing off-target effects. The effects of PBT on the antitumor immune response have only just begun to be described, and to our knowledge no studies to date have examined the effect of PBT as part of a combinatorial immunoradiotherapeutic strategy. Here, using a 2-tumor model of lung cancer in mice, we show that PBT in tandem with an anti-PD1 antibody substantially reduced growth in both irradiated and unirradiated tumors. This was accompanied by robust activation of the immune response, as evidenced by whole-tumor and single-cell RNA sequencing showing upregulation of a multitude of immune-related transcripts. This response was further significantly enhanced by the injection of the tumor to be irradiated with NBTXR3 nanoparticles. Tumors of mice treated with the triple combination exhibited increased infiltration and activation of cytotoxic immune cells. This triple combination eradicated both tumors in 37.5% of the treated mice and showed robust long-term immunity to cancer.

The role of tumor metabolism in modulating T-Cell activity and in optimizing immunotherapy.

Immunotherapy has revolutionized cancer treatment and revitalized efforts to harness the power of the immune system to combat a variety of cancer types more effectively. However, low clinical response rates and differences in outcomes due to variations in the immune landscape among patients with cancer continue to be major limitations to immunotherapy. Recent efforts to improve responses to immunotherapy have focused on targeting cellular metabolism, as the metabolic characteristics of cancer cells can directly influence the activity and metabolism of immune cells, particularly T cells. Although the metabolic pathways of various cancer cells and T cells have been extensively reviewed, the intersections among these pathways, and their potential use as targets for improving responses to immune-checkpoint blockade therapies, are not completely understood. This review focuses on the interplay between tumor metabolites and T-cell dysfunction as well as the relationship between several T-cell metabolic patterns and T-cell activity/function in tumor immunology. Understanding these relationships could offer new avenues for improving responses to immunotherapy on a metabolic basis.

Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer.

Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non-small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells-such as M2 tumor-associated macrophages (TAM)-thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti-PD-1-resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFß levels. The addition of anti-PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

NBTXR3 improves the efficacy of immunoradiotherapy combining nonfucosylated anti-CTLA4 in an anti-PD1 resistant lung cancer model.

The efficacy of immunoradiotherapy consisting of radiation therapy and immune checkpoint blockade relies on effectively promoting the systemic antitumor immune response's activation while simultaneously reducing local factors favoring immune suppression. We previously demonstrated that NBTXR3, a nanoparticle radioenhancer, significantly improved immune responses in a murine anti-PD1-resistant metastatic lung cancer model. We hypothesize that radioactivated-NBTXR3 addition to anti-PD1 and a second-generation anti-CTLA4 could improve treatment effectiveness. To test this hypothesis, we inoculated mice with 344SQR cells in the right and left legs to establish primary and secondary tumors. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by three fractions of 12 Gy radiation on days 8, 9, and 10. The secondary tumors received two fractions of 1Gy radiation on days 13 and 14. Multiple rounds of anti-PD1, anti-CTLA4 or nonfucosylated anti-CTLA4 were given to the mice. Immune profiling of the tumors revealed that the combination of NBTXR3 with immunoradiotherapy significantly upregulated the activities of a wide range of antitumor immune pathways and reduced the abundance of regulatory suppressor T cells. This combination effectively eradicated the primary and secondary tumors and increased animal survival to 75%. Remarkably, previously treated with NBTXR3-containing treatment, the survivor mice exhibited a long-lasting antitumor memory immune response. This data provides compelling evidence of the efficacy of NBTXR3 to synergize with the immunoradiotherapy approach when combined with an anti-PD1 and multiple checkpoints such as a second generation anti-CTLA4 and show the potential for clinical uses of antitumor immunomodulatory effects of NBTXR3.

Pulsed radiotherapy to mitigate high tumor burden and generate immune memory.

Radiation therapy (XRT) has a well-established role in cancer treatment. Given the encouraging results on immunostimulatory effects, radiation has been increasingly used with immune-check-point inhibitors in metastatic disease, especially when immunotherapy fails due to tumor immune evasion. We hypothesized that using high-dose stereotactic radiation in cycles (pulses) would increase T-cell priming and repertoire with each pulse and build immune memory in an incremental manner. To prove this hypothesis, we studied the combination of anti-CTLA-4 and Pulsed radiation therapy in our 344SQ non-small cell lung adenocarcinoma murine model. Primary and secondary tumors were bilaterally implanted in 129Sv/Ev mice. In the Pulsed XRT group, both primary and secondary tumors received 12Gyx2 radiation one week apart, and blood was collected seven days afterwards for TCR repertoire analysis. As for the delayed-Pulse group, primary tumors received 12Gyx2, and after a window of two weeks, the secondary tumors received 12Gyx2. Blood was collected seven days after the second cycle of radiation. The immunotherapy backbone for both groups was anti-CTLA-4 antibody to help with priming. Treatment with Pulsed XRT + anti-CTLA-4 led to significantly improved survival and resulted in a delayed tumor growth, where we observed enhanced antitumor efficacy at primary tumor sites beyond XRT + anti-CTLA-4 treatment group. More importantly, Pulsed XRT treatment led to increased CD4+ effector memory compared to single-cycle XRT. Pulsed XRT demonstrated superior efficacy to XRT in driving antitumor effects that were largely dependent on CD4+ T cells and partially dependent on CD8+ T cells. These results suggest that combinatorial strategies targeting multiple points of tumor immune evasion may lead to a robust and sustained antitumor response.

Combining a nanoparticle-mediated immunoradiotherapy with dual blockade of LAG3 and TIGIT improves the treatment efficacy in anti-PD1 resistant lung cancer.

BACKGROUND: While improvements in immunoradiotherapy have significantly improved outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to immunoradiotherapy is that immune cells may be suppressed via the myriad of different immune checkpoint receptors. Therefore, simultaneous blockade of multiple immune checkpoint receptors may enhance the treatment efficacy of immunoradiotherapy. METHODS: We combined NBTXR3-enhanced localized radiation with the simultaneous blockade of three different checkpoint receptors: PD1, LAG3, and TIGIT, and tested the treatment efficacy in an anti-PD1-resistant lung cancer model in mice. 129 Sv/Ev mice were inoculated with fifty thousand αPD1-resistant 344SQR cells in the right leg on day 0 to establish primary tumors and with the same number of cells in the left leg on day 4 to establish the secondary tumors. NBTXR3 was intratumorally injected into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 µg), αLAG3 (200 µg), and αTIGIT (200 µg) were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. RESULTS: This nanoparticle-mediated combination therapy is effective at controlling the growth of irradiated and distant unirradiated tumors, enhancing animal survival, and is the only one that led to the destruction of both tumors in approximately 30% of the treated mice. Corresponding with this improved response is robust activation of the immune response, as manifested by increased numbers of immune cells along with a transcriptional signature of both innate and adaptive immunity within the tumor. Furthermore, mice treated with this combinatorial therapy display immunological memory response when rechallenged by the same cancer cells, preventing tumor engraftment. CONCLUSION: Our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radiotherapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients.

A radioenhancing nanoparticle mediated immunoradiation improves survival and generates long-term antitumor immune memory in an anti-PD1-resistant murine lung cancer model.

BACKGROUND: Combining radiotherapy with PD1 blockade has had impressive antitumor effects in preclinical models of metastatic lung cancer, although anti-PD1 resistance remains problematic. Here, we report results from a triple-combination therapy in which NBTXR3, a clinically approved nanoparticle radioenhancer, is combined with high-dose radiation (HDXRT) to a primary tumor plus low-dose radiation (LDXRT) to a secondary tumor along with checkpoint blockade in a mouse model of anti-PD1-resistant metastatic lung cancer. METHODS: Mice were inoculated with 344SQR cells in the right legs on day 0 (primary tumor) and the left legs on day 3 (secondary tumor). Immune checkpoint inhibitors (ICIs), including anti-PD1 (200 µg) and anti-CTLA4 (100 µg) were given intraperitoneally. Primary tumors were injected with NBTXR3 on day 6 and irradiated with 12-Gy (HDXRT) on days 7, 8, and 9; secondary tumors were irradiated with 1-Gy (LDXRT) on days 12 and 13. The survivor mice at day 178 were rechallenged with 344SQR cells and tumor growth monitored thereafter. RESULTS: NBTXR3 + HDXRT + LDXRT + ICIs had significant antitumor effects against both primary and secondary tumors, improving the survival rate from 0 to 50%. Immune profiling of the secondary tumors revealed that NBTXR3 + HDXRT + LDXRT increased CD8 T-cell infiltration and decreased the number of regulatory T (Treg) cells. Finally, none of the re-challenged mice developed tumors, and they had higher percentages of CD4 memory T cells and CD4 and CD8 T cells in both blood and spleen relative to untreated mice. CONCLUSIONS: NBTXR3 nanoparticle in combination with radioimmunotherapy significantly improves anti-PD1 resistant lung tumor control via promoting antitumor immune response.

Radiation Therapy Enhanced by NBTXR3 Nanoparticles Overcomes Anti-PD1 Resistance and Evokes Abscopal Effects.

PURPOSE: Radiation combined with PD1 blockade offers significant treatment benefits in several tumor types; however, anti-PD1 resistance precludes such benefits in many cases. Here we attempted to overcome anti-PD1 resistance by combining localized radiation with a radioenhancing nanoparticle (NBTXR3) and systemic anti-PD1 treatment to achieve abscopal effects in an anti-PD1-resistant mouse model of lung cancer. METHODS AND MATERIALS: Female 129Sv/Ev mice were inoculated with 344SQ anti-PD1-resistant (344SQR) or anti-PD1-sensitive (344SQP) metastatic lung cancer cells in the right leg on day 0 ("primary" tumor) and the left leg on day 4 ("secondary" tumor). Primary tumors were injected intratumorally with NBTXR3 on day 7 and were irradiated with 12 Gy on days 8, 9, and 10. Mice were given 6 intraperitoneal injections of anti-PD1. T cell receptor repertoire was analyzed in tumor samples with RNA sequencing, infiltration of CD8 T cells with immunohistochemical staining, and activities of various immune pathways with NanoString analysis. RESULTS: The triple combination of NBTXR3 with localized radiation and systemic anti-PD1 significantly delayed the growth of both irradiated and unirradiated tumors in both 344SQP and 344SQR tumor models. NBTXR3 remodeled the immune microenvironment of unirradiated tumors by triggering the activation of various immune pathways, increasing the number of CD8+ T cells, and modifying the T cell receptor repertoire in the 344SQR tumor model. CONCLUSIONS: The ability of NBTXR3 to evoke significant abscopal effects in both anti-PD1-sensitive and anti-PD1-resistant lung cancers could open the possibility of its use for treating patients with metastatic lung cancer regardless of sensitivity (or resistance) to immunotherapies.

Addition of TLR9 agonist immunotherapy to radiation improves systemic antitumor activity.

Radiotherapy (RT) has been used to control tumors by physically damaging DNA and inducing apoptosis; it also promotes antitumor immune responses via neoantigens release and augmenting immune-oncology agents to elicit systemic response. Tumor regression after RT can recruit inflammatory cells, such as tumor-associated macrophages and CD11b+ myeloid cell populations, a major subset of which may actually be immunosuppressive. However, these inflammatory cells also express Toll-like receptors (TLRs) that can be stimulated to reverse suppressive characteristics and promote systemic antitumor outcomes. Here, we investigated the effects of adding CMP-001, a CpG-A oligodeoxynucleotide TLR9 agonist delivered in a virus-like particle (VLP), to RT in two murine models (344SQ metastatic lung adenocarcinoma and CT26 colon carcinoma). High-dose RT (12Gy x 3 fractions) significantly increased the percentages of plasmacytoid dendritic cells within the tumor islets 3- and 5-days post-RT; adding CMP-001 after RT also enhanced adaptive immunity by increasing the proportion of CD4+ and CD8+ T cells. RT plus CMP-001-mediated activation of the immune system led to significant inhibition of tumor growth at both primary and abscopal tumor sites, thereby suggesting a new combinatorial treatment strategy for systemic disease.

Low-dose radiation treatment enhances systemic antitumor immune responses by overcoming the inhibitory stroma.

BACKGROUND: Despite some successes with checkpoint inhibitors for treating cancer, most patients remain refractory to treatment, possibly due to the inhibitory nature of the tumor stroma that impedes the function and entry of effector cells. We devised a new technique of combining immunotherapy with radiotherapy (XRT), more specifically low-dose XRT, to overcome the stroma and maximize systemic outcomes. METHODS: We bilaterally established 344SQ lung adenocarcinoma tumors in 129Sv/Ev mice. Primary and secondary tumors were irradiated with either high-dose or low-dose of XRT with systemic anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte associated protein 4 administration. Survival and tumor growth were monitored for the various groups, and secondary tumors were phenotyped by flow cytometry for immune populations. Tumor growth factor-beta (TGF-ß) cytokine levels were assessed locally after low-dose XRT, and specific immune-cell depletion experiments were conducted to identify the major contributors to the observed systemic antitumor effect. RESULTS: Through our preclinical and clinical studies, we observed that when tumor burden was high, there was a necessity of combining high-dose XRT to 'prime' T cells at the primary tumor site, with low-dose XRT directed to secondary (metastatic) tumors to 'modulate the stroma'. Low-dose XRT improved the antitumor outcomes of checkpoint inhibitors by favoring M1 macrophage polarization, enhancing natural killer (NK) cell infiltration, and reducing TGF-ß levels. Depletion of CD4+ T cells and NK cells abrogated the observed antitumor effect. CONCLUSION: Our data extend the benefits of low-dose XRT to reprogram the tumor environment and improve the infiltration and function of effector immune cells into secondary tumors.

Author Correction: Bone morphogenetic protein 7 promotes resistance to immunotherapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bone morphogenetic protein 7 promotes resistance to immunotherapy.

Immunotherapies revolutionized cancer treatment by harnessing the immune system to target cancer cells. However, most patients are resistant to immunotherapies and the mechanisms underlying this resistant is still poorly understood. Here, we report that overexpression of BMP7, a member of the TGFB superfamily, represents a mechanism for resistance to anti-PD1 therapy in preclinical models and in patients with disease progression while on immunotherapies. BMP7 secreted by tumor cells acts on macrophages and CD4+ T cells in the tumor microenvironment, inhibiting MAPK14 expression and impairing pro-inflammatory responses. Knockdown of BMP7 or its neutralization via follistatin in combination with anti-PD1 re-sensitizes resistant tumors to immunotherapies. Thus, we identify the BMP7 signaling pathway as a potential immunotherapeutic target in cancer.

Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive malignancy with limited treatment options. We previously found that PARP is overexpressed in SCLC and that targeting PARP reduces cell line and tumor growth in preclinical models. However, SCLC cell lines with PI3K/mTOR pathway activation were relatively less sensitive to PARP inhibition. In this study, we investigated the proteomic changes in PI3K/mTOR and other pathways that occur following PAPR inhibition and/or knockdown in vitro and in vivo. Using reverse-phase protein array, we found the proteins most significantly upregulated following treatment with the PARP inhibitors olaparib and rucaparib were in the PI3K/mTOR pathway (p-mTOR, p-AKT, and pS6) (p≤0.02). Furthermore, amongst the most significantly down-regulated proteins were LKB1 and its targets AMPK and TSC, which negatively regulate the PI3K pathway (p≤0.042). Following PARP knockdown in cell lines, phosphorylated mTOR, AKT and S6 were elevated and LKB1 signaling was diminished. Global ATP concentrations increased following PARP inhibition (p≤0.02) leading us to hypothesize that the observed increased PI3K/mTOR pathway activation following PARP inhibition results from decreased ATP usage and a subsequent decrease in stress response signaling via LKB1. Based on these results, we then investigated whether co-targeting with a PARP and PI3K inhibitor (BKM-120) would work better than either single agent alone. A majority of SCLC cell lines were sensitive to BKM-120 at clinically achievable doses, and cMYC expression was the strongest biomarker of response. At clinically achievable doses of talazoparib (the most potent PARP inhibitor in SCLC clinical testing) and BKM-120, an additive effect was observed in vitro. When tested in two SCLC animal models, a greater than additive interaction was seen (p≤0.008). The data presented here suggest that combining PARP and PI3K inhibitors enhances the effect of either agent alone in preclinical models of SCLC, warranting further investigation of such combinations in SCLC patients.

Proteomic profiling identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets including PARP1.

UNLABELLED: Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLCs and NSCLCs. SCLCs showed lower levels of several receptor tyrosine kinases and decreased activation of phosphoinositide 3-kinase (PI3K) and Ras/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) pathways but significantly increased levels of E2F1-regulated factors including enhancer of zeste homolog 2 (EZH2), thymidylate synthase, apoptosis mediators, and DNA repair proteins. In addition, PARP1, a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLCs. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than were NSCLCs, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy. SIGNIFICANCE: SCLC is a highly lethal cancer with a 5-year survival rate of less than 10%. To date, no molecularly targeted agents have prolonged survival in patients with SCLCs. As a step toward identifying new targets, we systematically profiled SCLCs with a focus on therapeutically relevant signaling pathways. Our data reveal fundamental differences in the patterns of pathway activation in SCLCs and NSCLCs and identify several potential therapeutic targets for SCLCs, including PARP1 and EZH2. On the basis of these results, clinical studies evaluating PARP and EZH2 inhibition, together with chemotherapy or other agents, warrant further investigation.