Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are commonly used to treat non-small cell lung cancers with EGFR mutations, but drug resistance often emerges. Intratumor heterogeneity is a known cause of targeted therapy resistance and is considered a major factor in treatment failure. This study identifies clones of EGFR-mutant non-small cell lung tumors expressing low levels of both wild-type and mutant EGFR protein. These EGFR-low cells are intrinsically more tolerant to EGFR inhibitors, more invasive, and exhibit an epithelial-to-mesenchymal-like phenotype compared to their EGFR-high counterparts. The EGFR-low cells secrete Transforming growth factor beta (TGFβ) family cytokines, leading to increased recruitment of cancer-associated fibroblasts and immune suppression, thus contributing to the drug-tolerant tumor microenvironment. Notably, pharmacological induction of EGFR using epigenetic inhibitors sensitizes the resistant cells to EGFR inhibition. These findings suggest that intrinsic drug resistance can be prevented or reversed using combination therapies.

Despite small cell lung cancers (SCLCs) having a high mutational burden, programmed death-ligand 1 (PD-L1) immunotherapy only modestly increases survival. A subset of SCLCs that lose their ASCL1 neuroendocrine phenotype and restore innate immune signaling (termed the "inflammatory" subtype) have durable responses to PD-L1. Some SCLCs are highly sensitive to Aurora kinase inhibitors, but early-phase trials show short-lived responses, suggesting effective therapeutic combinations are needed to increase their durability. Using immunocompetent SCLC genetically engineered mouse models (GEMMs) and syngeneic xenografts, we show durable efficacy with the combination of a highly specific Aurora A kinase inhibitor (LSN3321213) and PD-L1. LSN3321213 causes accumulation of tumor cells in mitosis with lower ASCL1 expression and higher expression of interferon target genes and antigen-presentation genes mimicking the inflammatory subtype in a cell-cycle-dependent manner. These data demonstrate that inflammatory gene expression is restored in mitosis in SCLC, which can be exploited by Aurora A kinase inhibition.

Supplementary Data from A Novel HER2-Selective Kinase Inhibitor Is Effective in HER2 Mutant and Amplified Non–Small Cell Lung Cancer

Supplementary Methods and Legends

Abstract In-frame insertions in exon 20 of HER2 are the most common HER2 mutations in patients with non–small cell lung cancer (NSCLC), a disease in which approved EGFR/HER2 tyrosine kinase inhibitors (TKI) display poor efficiency and undesirable side effects due to their strong inhibition of wild-type (WT) EGFR. Here, we report a HER2-selective covalent TKI, JBJ-08–178–01, that targets multiple HER2 activating mutations, including exon 20 insertions as well as amplification. JBJ-08–178–01 displayed strong selectivity toward HER2 mutants over WT EGFR compared with other EGFR/HER2 TKIs. Determination of the crystal structure of HER2 in complex with JBJ-08–178–01 suggests that an interaction between the inhibitor and Ser783 may be responsible for HER2 selectivity. The compound showed strong antitumoral activity in HER2-mutant or amplified cancers in vitro and in vivo. Treatment with JBJ-08–178–01 also led to a reduction in total HER2 by promoting proteasomal degradation of the receptor. Taken together, the dual activity of JBJ-08–178–01 as a selective inhibitor and destabilizer of HER2 represents a combination that may lead to better efficacy and tolerance in patients with NSCLC harboring HER2 genetic alterations or amplification. Significance: This study describes unique mechanisms of action of a new mutant-selective HER2 kinase inhibitor that reduces both kinase activity and protein levels of HER2 in lung cancer.

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

Malignant pleural mesothelioma (MPM) is an aggressive cancer defined by loss-of-function mutations with few therapeutic options. We examined the contribution of the transcription factor Signal transducer and activator of transcription 3 (STAT3) to cell growth and gene expression in preclinical models of MPM. STAT3 is activated in a variety of tumors and is thought to be required for the maintenance of cancer stem cells. Targeting STAT3 using specific small hairpin RNAs (shRNAs) or with the pharmacologic inhibitors atovaquone or pyrimethamine efficiently reduced cell growth in established cell lines and primary-derived lines while showing minimal effects in nontransformed LP9 mesothelial cells. Moreover, atovaquone significantly reduced viability and tumor growth in microfluidic cultures of primary MPM as well as in an in vivo xenotransplant model. Biological changes were linked to modulation of gene expression associated with STAT3 signaling, including cell cycle progression and altered p53 response. Reflecting the role of STAT3 in inducing localized immune suppression, using both atovaquone and pyrimethamine resulted in the modulation of immunoregulatory genes predicted to enhance an immune response, including upregulation of ICOSLG (Inducible T-Cell Costimulator Ligand or B7H2). Thus, our data strongly support a role for STAT3 inhibitors as anti-MPM therapeutics.

T-cells are critical effector cells of cancer immunotherapies, but little is known about T-cell gene expression programs in diffuse gliomas. We leveraged single-cell RNA-seq to chart the gene expression and clonal landscape of tumor-infiltrating T-cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. Our analysis revealed subsets of T-cells that expressed several NK-cell receptors, in particular the inhibitory CD161 receptor (KLRB1 gene). KLRB1 was overexpressed by clonally expanded CD8 T-cells, and larger populations of T-cells expressed CD161 than PD-1. The CLEC2D ligand of CD161 was expressed by malignant cells and myeloid cells, and inactivation of KLRB1 enhanced anti-tumor T-cell function. KLRB1 was also expressed by substantial T-cell populations in multiple other human cancers. CD161 and other NK-cell receptors expressed by T-cells represent opportunities for immunotherapy of diffuse gliomas and other human cancers.

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in EGFR-mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

MYD88 mutations are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of Diffuse Large B-cell Lymphoma (ABC DLBCL). Mutated MYD88 activates BTK, and triggers downstream pro-survival signaling that includes NF-kB and ERK (Yang et al, Blood 2013; Blood 2016). ERK related signaling triggers inflammatory cytokine production including IL-6 and IL-10 (Chen et al, Blood 2016). Ibrutinib covalently binds to BTK Cys481 and inactivates BTK and downstream NF-kB and ERK signaling. Ibrutinib is approved for the treatment of WM and is associated with high overall response rates (>90%) and long term progression free survival in WM though intolerance to therapy, as well as resistance related to acquired BTK Cys481 mutations frequently leads to treatment discontinuation. We therefore investigated a novel, non-covalent BTK-inhibitor, pirtobrutinib that binds to BTK at non-Cys481 amino acids (G473-K483). Pirtobrutinib showed highly selective anti-proliferative activity against MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8 and HBL-1) versus MYD88 wild-type (OCI-Ly7, OCI-Ly19, Ramos, and RPMI-8226) cells, with marked apoptotic effect exhibited against primary MYD88 mutated WM cells at pharmacologically achievable levels (100-500 nM). Importantly, pirtobrutinib blocked BTK activity and overcame ibrutinib resistance in BCWM.1 WM and TMD-8 ABC DLBCL cells transduced to express both wild-type and mutated BTK (BTK Cys481Ser) with similar efficacy. The downstream signaling consequences of pirtobrutinib in vector only, wild-type and mutant BTK Cys481 expressing BCWM.1, MWCL-1, TMD-8 and HBL-1 cells was also examined. Treatment of vector only and wild-type BTK Cys481 expressing WM and ABC DLBCL cells with ibrutinib or pirtobrutinib abrogated both p-BTK and p-ERK signaling. In contrast, only pirtobrutinib blocked p-BTK and p-ERK signaling in mutant BTK Cys481 expressing WM and ABC DLBCL cells. In previous studies, we showed that inflammatory cytokine production that included IL-6 and IL-10 driven by ERK triggered ibrutinib resistance in wild-type BTK Cys481 MYD88 mutated lymphoma cells co-cultured with their mutated BTK expressing counterparts (Chen et al, BLOOD 2018). ERK-driven cytokine resistance to ibrutinib was postulated to explain how disease progression occurs in patients with modest variant expression of mutated BTK Cys481 (Woyach et al, JCO 2017; Xu et al, Blood 2017). Co-culture of BTK Cys481 mutated expressing TMD-8 cells with wild-type BTK expressing TMD-8 cells triggered resistance of the latter to ibrutinib. Treatment with pirtobrutinib blocked IL-6 and IL-10 production and overcame the protective effects conferred by BTK Cys481 mutated TMD-8 cells in these experiments. Lastly, oral administration of pirtobrutinib blocked p-BTK and p-ERK in BTK Cys481 mutated TMD-8 tumors xenografted in mice. Our findings therefore show that pirtobrutinib inhibits growth of MYD88 mutated lymphoma cells in a highly selective manner and can trigger apoptosis of primary WM patient BM lymphoplasmacytic cells at levels comparable to ibrutinib. Moreover, pirtobrutinib effectively blocked mutated BTK Cys481 driven BTK and ERK1/2 activation and produced similar cellular efficacy in both BTK wild-type and BTK Cys481 mutated cells. Pirtobrutinib also blocked the protective effect conferred to BTK wild-type cells through paracrine cytokines released by BTK Cys481Ser expressing cells. Lastly, pirtobrutinib blocked BTK and ERK1/2 activation in TMD8-BTK Cys481Ser xenografted mice. The findings support the development of pirtobrutinib in MYD88 driven lymphomas, including those resistant to ibrutinib on the bases of BTK Cys481 mutations.

Abstract Purpose: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy. Experimental Design: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors. Results: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti–PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti–PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30–3.68; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23–3.16; P = 0.005]. Conclusions: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.

Abstract Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. Significance: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection. This article is highlighted in the In This Issue feature, p. 1861

BackgroundRecently, TIGIT+PD1 blockade was shown to confer additive survival benefits in orthotopic glioblastoma and colon cancer mouse models1 2—notably, this included experiments3 that used the 1G9 TIGIT monoclonal antibody (mAb) that has potentially agonistic effects.1 Herein we investigated the TIGIT/CD226/CD155 axis and effects of combining clinically analogous PD-1/PD-L1 mAbs with TIGIT-targeting 1G9 mAb in murine glioblastoma models.MethodsThe overall survival (OS) associated with TIGIT (non-depleting IG9; 200μg every 3days for 4 doses),1 PD-1 (8H3; initial 500μg followed by 250μg every 3days for 7 doses), PD-L1 (6A2; initial 500μg followed by 250μg every 3days for 7 doses), and/or IgG mAbs was assessed in immunocompetent C57BL/6 albino mice intracranially implanted with syngeneic GL261-luc2 or CT2A-luc.4 5 The roles of T cells and NK cells were examined using depletion with CD8a, CD4, or NK1.1 mAbs. Expression of TIGIT/CD226/CD155 and PD-1/PD-L1/PD-L2 by tumor and tumor-infiltrating immune cells was evaluated using flow cytometry and RT-qPCR.ResultsIn vitro, GL261-luc2 and CT2A-luc tumor cells moderately expressed PD-L1, PD-1, and TIGIT; but strongly expressed TIGIT’s inhibitory ligand CD155.(Figure1A-B) Ex vivo, >83% of CD8+ and CD4+TILs in GL261-luc2 co-expressed TIGIT+/CD226+: ≥2x the proportions in CT2A-luc.(Figure 2A) CD155 and PD-L1 were highly co-expressed on tumor-infiltrating macrophages: greater in GL261-luc2 than CT2A-luc tumors.(Figure 2B)In GL261-luc2 mice, anti-TIGIT monotherapy displayed minimal OS improvement; whereas anti-PD-1 and anti-PD-L1 monotherapies demonstrated robust OS responses.(Figure 3A) Adding anti-TIGIT to PD-1/PD-L1 blockade demonstrated synergism with anti-PD-1. Anti-TIGIT plus anti-PD-1 displayed nominally improved OS in CT2A-luc compared to anti-PD-1 monotherapy (p=0.11).(Figure 3B).Given robust T-cell expression of TIGIT and PD-1, we examined how CD4+ or CD8+ depletion affected responses in GL261-luc2 mice: depletion completely abrogated anti-PD-1’s benefits.(Figure 4) Although CD4/CD8 depletion also reduced anti-TIGIT+anti-PD1’s efficacy, the resulting OS matched that of non-depleted anti-PD-1 monotherapy. Additionally, NK cell depletion had no effect on anti-TIGIT+anti-PD1’s efficacy.Abstract 256 Figure 1TIGIT/CD155 axis and PD-1/PD-L1/PD-L2 axis expression in murine glioblastoma model tumor cells. Protein and RNA expression of the TIGIT and PD-1 immune checkpoints — and their ligands CD155 and PDL-1/PD-L2 respectively — on GL261-luc2 and CT2A-luc tumor cells using (A) flow cytometry (blue=samples, red=unstained controls) and (B) RT-qPCR (grey=GL261-luc2, black=CT2A-luc). Pdcd1 encodes PD-1, Cd274 encodes PD-L1, Pvr encodes CD155Abstract 256 Figure 2TIGIT/CD155 axis and PD-1/PD-L1/PD-L2 axis expression in murine glioblastoma model tumor-infiltrating immune cells. (A) Flow cytometry analysis of protein expression for TIGIT (y-axis) and CD226 (x-axis; a competitor of TIGIT) on CD8+, CD4+/FOXP3+ Treg, and CD4+/FOXP3- Teff tumor-infiltrating lymphocytes (TILs) from GL261-luc2 and CT2A-luc tumor-bearing mice. (B) Flow cytometry analysis of protein expression for CD155 (TIGIT’s ligand; bottom) and PD-L1 (PD-1’s ligand; top) on tumor-infiltrating myeloid populations from GL261-luc2 and CT2A-luc tumor-bearing mice. Mean fluorescent intensity (MFI) was compared between cell lines, *indicates statistical significance. Myeloid populations included CD45+/CD11b+/CD11c+/F4-80+ macrophages, CD11b+/CD11c+ and CD11b-/CD11c+ dendritic cells (DCs), CD11b+/CD11c-/Ly6C+/Ly6G- monocytes, CD11b+/CD11c-/Ly6Cmid/Ly6G+ granulocytes, and CD45dim/CX3CR1+ microglia. Tumors were dissociated and leukocytes were enriched for using Percoll gradient. n=5 mice per group.Abstract 256 Figure 3The survival associated with TIGIT-targeting mAb therapy with/without clinically-analogous PD-1/PD-L1 blockade. (A) Kaplan-Meier estimated overall survival (measured from day of intracranial tumor implantation) of GL261-luc2 mice treated with TIGIT (1G9), PD-1 (8H3), PD-L1 (6A2), TIGIT + PD-1, TIGIT + PD-L1 mAbs, or IgG control. (B) A) Kaplan-Meier estimated overall survival (measured from day of intracranial tumor implantation) of CT2A-luc mice treated with TIGIT (1G9), PD-1 (8H3), TIGIT + PD-1 mAbs, or IgG control. For both experiments, all treatments were started on day 6 following implantation, with the following dosing: anti-TIGIT was given as 200μg every 3days for 4 doses. Both anti-PD-1 and anti-PD-L1 were given as an initial 500μg dose followed by 250μg every 3days for 7 doses.4 The n per group and number at risk table is included underneath each graph, along with the corresponding Cox regression analysis. Treatment groups with significantly different OS from the combination TIGIT + PD-1 combination-treated reference group were highlighted in yellow. HR = hazard ratio, CI = confidence interval.Abstract 256 Figure 4How depletion of CD8+ T cells, CD4+ T cells, or NK cells affects survival associated with TIGIT-targeting mAb therapy with/without clinically-analogous PD-1 blockade. Kaplan-Meier estimated overall survival (measured from day of intracranial tumor implantation) of GL261-luc2 mice treated with TIGIT (1G9), PD-1 (8H3), TIGIT + PD-1 mAbs, or IgG control; and compared to groups that additional had CD8+, CD4+, or NK1.1+ antibody-based depletion. The treatment and dosing characteristics were the same as Figure 3. The n per group and number at risk table is included underneath the graph, along with the corresponding Cox regression analysis. Treatment groups with significantly different OS from the combination TIGIT + PD-1 combination-treated reference group were highlighted in yellow. HR = hazard ratio, CI = confidence interval.ConclusionsOur results recapitulate published findings regarding the synergistic benefits of combining TIGIT 1G9 mAb with anti-PD-1 using the clinically-relevant 8H3 mAb in syngeneic mouse glioblastoma, and extend those findings to anti-TIGIT+anti-PDL1 combinations. TIGIT/CD226 was highly co-expressed by immuno-responsive GL261-luc2’s tumor-infiltrating lymphocytes (TILs); wheres CD155/PD-L1 expression predominated in tumor-infiltrating myeloid cells. Depletion of CD8+ or CD4+ TILs modestly reduced anti-TIGIT+anti-PD1’s efficacy—suggesting a mechanism that is at least partially independent of T (and NK) cells. Our preliminary results suggest a complex interplay between TIGIT/CD226/CD155 and PD-1/PD-L1/PD-L2 axes in tumors and their microenvironmental constituents that warrants further investigation; plus, careful consideration of antibody clones’ functionality is necessary for designing immunotherapy combinations.AcknowledgementsWe gratefully acknowledge the support of the The Jennifer Oppenheimer Cancer Research Initiative; The Ben and Catherine Ivy Foundation; Hope It’s A Beach Thing; and the Pan Mass Challenge (Erica’s Entourage and CRUS11TOUR), and the NCI (P01CA236749; K12CA090354).ReferencesDixon KO, Schorer M, Nevin J, Etminan Y, Amoozgar Z, Kondo T, Kurtulus S, Kassam N, Sobel RA, Fukumura D, Jain RK, Anderson AC, Kuchroo VK, Joller N. Functional anti-TIGIT antibodies regulate development of autoimmunity and antitumor immunity. J Immunol 2018 April 15;200(8):3000–3007.Hung AL, Maxwell R, Theodros D, Belcaid Z, Mathios D, Luksik AS, Kim E, Wu A, Xia Y, Garzon-Muvdi T, Jackson C, Ye X, Tyler B, Selby M, Korman A, Barnhart B, Park SM, Youn JI, Chowdhury T, Park CK, Brem H, Pardoll DM, Lim M. TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM. Oncoimmunology 2018 May 24;7(8):e1466769.Raphael I, Kumar R, McCarl LH, Shoger K, Wang L, Sandlesh P, Sneiderman CT, Allen J, Zhai S, Campagna ML, Foster A, Bruno TC, Agnihotri S, Hu B, Castro BA, Lieberman FS, Broniscer A, Diaz AA, Amankulor NM, Rajasundaram D, Pollack IF, Kohanbash G. TIGIT and PD-1 immune checkpoint pathways are associated with patient outcome and anti-tumor immunity in glioblastoma. Front Immunol 2021 May 7;12:637146.Reardon DA, Gokhale PC, Klein SR, Ligon KL, Rodig SJ, Ramkissoon SH, Jones KL, Conway AS, Liao X, Zhou J, Wen PY, Van Den Abbeele AD, Hodi FS, Qin L, Kohl NE, Sharpe AH, Dranoff G, Freeman GJ. Glioblastoma eradication following immune checkpoint blockade in an orthotopic, immunocompetent model. Cancer Immunol Res 2016 February;4(2):124–35. Iorgulescu JB, Gokhale PC, Speranza MC, Eschle BK, Poitras MJ, Wilkens MK, Soroko KM, Chhoeu C, Knott A, Gao Y, Lim-Fat MJ, Baker GJ, Bonal DM, Nguyen QD, Grant GRL, Ligon KL, Sorger PK, Chiocca EA, Anderson AC, Kirschmeier PT, Sharpe AH, Freeman GJ, Reardon DA. Concurrent dexamethasone limits the clinical benefit of immune checkpoint blockade in glioblastoma. Clin Cancer Res 2021 January 1;27(1):276–287.

Background Increasing data indicate that corticosteroids can exert a detrimental effect on immunotherapy for oncology patients. Dexamethasone, a uniquely potent corticosteroid, is frequently administered to brain tumor patients to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in glioblastoma patients – particularly in the context of immunotherapy. Methods We evaluated the dose-dependent effects of dexamethasone when administered with PD-1 blockade and/or radiotherapy on survival and tumor response in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A glioblastoma tumors. The immune microenvironment was comprehensively profiled using flow cytometry analysis. Clinically, the effect of dexamethasone on survival was evaluated in 181 IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors using multivariable Cox regression. Results Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner (figure 1). Concurrent dexamethasone also abrogated survival following anti-PD-1 with or without radiotherapy in immunoresistant CT-2A models (figure 2). Dexamethasone decreased T lymphocyte numbers (figure 3) by increasing apoptosis (figure 4), in addition to decreasing lymphocyte functional capacity (figure 3C/D). Myeloid and NK cell populations were also generally reduced by dexamethasone (figure 3). Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive IDH-wildtype glioblastoma patients treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment by relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival, regardless of dose (referent no dexamethasone; <2 mg HR 2.16, 95%CI: 1.30–3.68, p=0.003; ≥2 mg HR 1.97, 95%CI: 1.23–3.16, p=0.005; table 1 and figure 5). Conclusions We demonstrate that concurrent dexamethasone administration, even at a low dose, limits the therapeutic benefit of anti-PD-1 therapy both in mouse glioblastoma models and in a retrospective cohort of 181 IDH-wildtype glioblastoma patients. Mechanistically, dexamethasone decreased intratumoral T cells and systemic levels of T cells, natural killer cells, and myeloid cells, while qualitatively impairing lymphocyte function. The mechanism of T cell depletion included induction of apoptosis. These findings indicate that dexamethasone hinders both adaptive and innate immune responses, intratumorally and systemically, and that its administration should be carefully assessed among glioblastoma patients undergoing second-generation immunotherapy clinical trials. Our findings also have ramifications for brain metastasis patients where immune checkpoint inhibitors are part of standard-of-care management. Acknowledgements We thank Min Wu for assistance in generating CT-2A luciferase-transduced cells, and Drs. Geoffrey Young, Lei Qin, Xin Chen, and Jing Li for assistance in evaluation of patients' radiographic imaging. Ethics Approval Approved under DFCI Institutional Review Board protocol 10-417.

TET2 is among the most frequently mutated genes in hematopoietic malignancies. Inactivating mutations in TET2 are found in ∼30% of patients with myelodysplastic syndrome (MDS), ∼19% with de novo acute myeloid leukemia (AML), and ~15% with myeloproliferative neoplasm (MPN). TET2 mutations are also identified in a subset of individuals over 50 years of age with clonal hematopoiesis of indeterminate potential (CHIP), a condition that predisposes affected individuals to progression to myeloid malignancy and atherosclerotic heart disease with heart attack or stroke. TET2 mutations represent an early genetic lesion in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state of clonal dominance that predisposes to the acquisition of additional mutations. Thus, effective therapy for patients with TET2 mutations in HSPCs will require identifying drugs that are selectively lethal to TET2 mutant HSPCs but spare normal HSPCs, a property analogous to the widely studied genetic relationship called “synthetic lethality”. Using a tet2-mutant zebrafish model created in our laboratory, we screened for drugs from libraries of FDA-approved compounds and drugs in Phase I/II testing. We found that nuclear exporter XPO1 inhibitors, selinexor (KPT-330) and eltanexor (KPT-8602), were among the most promising, in that they are selectively lethal to HSPCs in tet2-mutant compared to wild-type fish. Moreover, we treated HSPCs of wild-type and Tet2-deficient mice with both selinexor and eltanexor in a methylcellulose colony formation assay. WT HSPCs lose their replating capacity at passage 3 (P3), while Tet2-mutant clones demonstrate aberrant sustained self-renewal capacity over multiple replatings. We found that both drugs selectively kill primary Tet2-mutant murine HSPCs and also block the aberrant self-renewal of these cells. By transplanting WT and Tet2-mutant CD45.2 donor cells into CD45.1 recipient mice, we monitored peripheral blood cells before and after treatment with selinexor or eltanexor to explore synthetic lethality of these drugs in vivo. Moreover, the human AML cell line K562 with TET2 homozygous mutations generated with CRISPR-Cas9, was more sensitive to selinexor and eltanexor than the parental cell line. To elucidate the mechanism behind the selective targeting of Tet2-mutant blood stem cells, we will use PRO-Seq and START-Seq methods to identify transcriptional elongation and initiation sites at single-nucleotide resolution. These preclinical studies must be done to show a therapeutic efficacy and mechanism of action of these new drugs before they can be translated to improve therapy of patients who have TET2 inactivating mutations. Citation Format: Nicole Prutsch, Chang-Bin Jing, Julia Etchin, Alla Berezovskaya, Hong Tiv, Michael J. Poitras, Prafulla Gokhale, Yosef Landesman, A. Thomas Look. Selective activity of XPO1 inhibitors in TET2-mutant myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2953.

Introduction: Non-small cell lung cancers harboring sensitizing gain-of-function mutations in the epidermal growth factor receptor (EGFR) are treated with small molecule EGFR kinase inhibitors including erlotinib and osimertinib. Unfortunately, the clinical efficacy of these inhibitors is limited by progression to drug resistance. Genomic amplification of the hepatocyte growth factor receptor (MET) is a prevalent mechanism of resistance to clinical EGFR inhibitors. EGFR mutant lung cancers with MET amplification are co-dependent on signaling through EGFR and MET kinases, whereby either oncogene can phosphorylate the EGFR family receptor ERBB3, activating downstream signaling and promoting survival. These EGFR/MET co-dependent cancers are characteristically resistant to treatment with individual drugs, but retain sensitivity to combination treatment with EGFR and MET inhibitors. Here, we describe three patient-derived EGFR mutant, MET amplified lung cancer models that exhibit a switch away from EGFR, to MET oncogene dependence. Methods: We established and characterized patient-derived lung cancer cell lines and xenograft models harboring concurrent EGFR activating mutations (EGFR L858R or EGFR Del19) and genomic MET copy number gain. Three patient-derived models—DFCI81, DFCI161, and DFCI307—were developed from pleural effusion and core biopsy specimens of patients whose tumors progressed on erlotinib. Clinical samples were immediately sorted and stably cultured in vitro or engrafted into immunodeficient mice. Results: DFCI81, DFCI161, and DFCI307 cell lines and xenograft models retained mutant EGFR expression, but exhibited resistance to clinical EGFR inhibitors and sensitivity to single-agent c-MET inhibitors, including crizotinib and savolitinib. Comparing our EGFR-mutant and MET-dependent cell lines to EGFR/MET-codependent models, we observed that EGFR expression was significantly reduced in our models compared to controls. Ectopic overexpression of EGFR Del19 and EGFR L858R in DFCI81 and DFCI161 cell lines, respectively, was sufficient to confer crizotinib resistance and induce EGFR/MET co-dependency. We demonstrated that MET-mediated ERBB3 phosphorylation drives downstream PI3K activation to promote cell proliferation and survival in DFCI81 and DFCI161 cells. In these contexts, ERBB3 reactivation by recombinant ligand treatment was sufficient to induce EGFR-mediated ERBB3 activation, conferring resistance to single-agent crizotinib treatment. Conclusions: We have identified and characterized three patient-derived models of treatment refractory EGFR mutant lung cancer that exhibit a switch to MET oncogene dependency. Clinically, we predict a subset of EGFR mutant, MET-dependent tumors exists, and can be identified de novo by a reduced EGFR to MET expression ratio: a potential biomarker predictive of sensitivity to single-agent MET inhibition. Citation Format: Pinar Ö. Eser, Raymond M. Paranal, Michael J. Poitras, Man Xu, Stephen Wang, Atsuko Ogino, Jihyun Choi, Pavlos Missios, Heidi M. Haikala, Jieun Son, Mika Lin, Masahiko Yanagita, Prafulla C. Gokhale, George Q. Daley, Pasi A. Jänne. Switch to MET oncogene dependence as a novel mechanism of drug resistance in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1732.