Spatial Mapping and Immunomodulatory Role of the OX40/OX40L Pathway in Human Non-Small Cell Lung Cancer.

PURPOSE: To evaluate the tissue distribution and clinical significance of OX40 and OX40L in human non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using multiplexed quantitative immunofluorescence, we conducted simultaneous and localized measurements of OX40 and OX40L proteins, major T-cell subsets, and conventional type 1 dendritic cells (cDC1) in 614 primary NSCLCs from three independent cohorts represented in tissue microarrays. We also measured OX40L protein in samples from a phase I clinical trial of intratumor administration of a lipid nanoparticle encapsulated mRNA encoding OX40L (mRNA-2416) in human solid tumors. Finally, we studied the OX40 pathway in 212 uterine/ovarian serous carcinomas. RESULTS: OX40 protein was expressed in approximately 90% of NSCLCs, and OX40L was detected in approximately 10% of cases. Increased expression of OX40 was associated with higher CD4+ and CD8+ T lymphocytes, as well as cDC1s. Elevated expression of OX40L was consistently associated with increased CD4+ tumor-infiltrating lymphocytes and longer overall survival. No association was found between OX40 or OX40L levels and oncogenic driver mutations in EGFR and KRAS in lung adenocarcinomas. Delivering OX40L mRNA using intratumor mRNA-2416 injection mediated increased local OX40L protein levels that was most prominent in a patient with ovarian serous carcinoma. Detectable OX40L protein levels were observed in 15% of primary uterine/ovarian serous malignancies and associated with longer survival. CONCLUSIONS: The OX40 pathway is expressed in a fraction of NSCLCs and is associated with a favorable immune contexture. Although OX40L is uncommonly expressed in NSCLC and serous malignancies, it is associated with better prognosis and can be introduced using exogenous mRNA.

The Combined Effect of FGFR Inhibition and PD-1 Blockade Promotes Tumor-Intrinsic Induction of Antitumor Immunity.

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs.

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

Lung stem cell self-renewal relies on BMI1-dependent control of expression at imprinted loci.

BMI1 is required for the self-renewal of stem cells in many tissues including the lung epithelial stem cells, Bronchioalveolar Stem Cells (BASCs). Imprinted genes, which exhibit expression from only the maternally or paternally inherited allele, are known to regulate developmental processes, but what their role is in adult cells remains a fundamental question. Many imprinted genes were derepressed in Bmi1 knockout mice, and knockdown of Cdkn1c (p57) and other imprinted genes partially rescued the self-renewal defect of Bmi1 mutant lung cells. Expression of p57 and other imprinted genes was required for lung cell self-renewal in culture and correlated with repair of lung epithelial cell injury in vivo. Our data suggest that BMI1-dependent regulation of expressed alleles at imprinted loci, distinct from imprinting per se, is required for control of lung stem cells. We anticipate that the regulation and function of imprinted genes is crucial for self-renewal in diverse adult tissue-specific stem cells.

Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase.

DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, we expressed a mutant version of Drosophila Cdt1 (Dup), which lacks the PCNA-binding PIP box (Dup(ΔPIP)) and which cannot be regulated by CRL4(Cdt2). Dup(ΔPIP) is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. Dup(ΔPIP) restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, Dup(ΔPIP) accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of Dup(ΔPIP) expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. We could not detect Dup hyperaccumulation using mutations in the CRL4(Cdt2) components Cul4 and Ddb1, likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain Dup(ΔPIP) activity in some endocycling cell types.

Bmi1 is critical for lung tumorigenesis and bronchioalveolar stem cell expansion.

Understanding the pathways that control epithelial carcinogenesis is vital to the development of effective treatments. The Polycomb group family member Bmi1 is overexpressed in numerous epithelial tumors, but its role in their development has not been established. We now show a key role for Bmi1 in lung adenocarcinoma. Whereas lung development occurs normally in Bmi1-deficient mice, loss of Bmi1 decreases the number and progression of lung tumors at a very early point in an oncogenic K-ras-initiated mouse model of lung cancer. This correlates with a defect in the ability of Bmi1-deficient putative bronchiolalveolar stem cells (BASCs) to proliferate in response to the oncogenic stimulus. Notably, in the absence of oncogenic K-ras, Bmi1-deficient BASCs show impaired proliferation and self-renewal capacity in culture and after lung injury in vivo. Abrogated lung cancer development and BASC self-renewal occur partially in a p19(ARF)-dependent manner. Our data suggest that Bmi1 deficiency suppresses tumor development by limiting the expansion potential of BASCs, the apparent lung cancer cells of origin. Because Bmi1 is elevated in additional tumor types, this suggests that Bmi1 plays a key role in regulating proliferation of both stem cells and tumor cells in diverse adult epithelial tissues.

Negative regulation of TSC1-TSC2 by mammalian D-type cyclins.

The metazoan cell cycle is driven by the timely and composite activities of cyclin-dependent kinases (CDKs). Among these, cyclin D- and cyclin E-dependent kinases phosphorylate the pRb family proteins during G(1) phase of the cell cycle and thereby advance cells beyond the restriction point. Increasing evidence suggests that cyclin D-dependent kinases might affect events other than Rb pathway-mediated entry into S phase, such as accumulation of cell mass. However, little is known about cyclin D activity toward Rb-independent pathway(s) or non-pRb substrates. In this article, we show that the tumor suppressor TSC2 is a cyclin D binding protein. Coexpression of cyclin D1-CDK4/6 in cultured cells leads to increased phosphorylation and decreased detection of both TSC2 and TSC1, and promotes the phosphorylation of the mTOR substrates, 4E-BP1 and S6K1, two key effectors of cell growth that are negatively regulated by the TSC1-TSC2 complex. At the cellular level, ectopic expression of cyclin D1 restores the cell size decrease caused by TSC1-TSC2 expression. Intriguingly, down-regulation of TSC proteins was also observed by the expression of a mutant cyclin D1 that is unable to bind to CDK4/6, or by the coexpression of cyclin D1 with either an INK4 inhibitor or with catalytically inactive CDK6, indicating that cyclin D may regulate TSC1-TSC2 independently of CDK4/6. Together, these observations suggest that mammalian D-type cyclins participate in cell growth control through negative regulation of TSC1-TSC2 function.