Perry et al. show that myeloid-targeted immunotherapy with a combination of anti-CD40 and CSF-1R inhibition synergistically induces a proinflammatory microenvironment that suppresses CPI-resistant tumors in a TNF-α– and IFN-γ–dependent manner.

Background Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib are effective for treating patients with EGFR mutant lung cancer; however, drug resistance inevitably emerges. Approaches to combine immunotherapies and targeted therapies to overcome or delay drug resistance have been hindered by limited knowledge of the effect of erlotinib on tumor-infiltrating immune cells. Methods Using mouse models, we studied the immunological profile of mutant EGFR-driven lung tumors before and after erlotinib treatment. Results We found that erlotinib triggered the recruitment of inflammatory T cells into the lungs and increased maturation of alveolar macrophages. Interestingly, this phenotype could be recapitulated by tumor regression mediated by deprivation of the EGFR oncogene indicating that tumor regression alone was sufficient for these immunostimulatory effects. We also found that further efforts to boost the function and abundance of inflammatory cells, by combining erlotinib treatment with anti-PD-1 and/or a CD40 agonist, did not improve survival in an EGFR-driven mouse model. Conclusions Our findings lay the foundation for understanding the effects of TKIs on the tumor microenvironment and highlight the importance of investigating targeted and immuno-therapy combination strategies to treat EGFR mutant lung cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0643-8) contains supplementary material, which is available to authorized users.

◥Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFR L858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance in vivo.Significance: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance.

Osimertinib, a mutant‐specific third generation EGFR TKI, is emerging as the preferred first‐line therapy for EGFR mutant lung cancer. Despite initial responses in patients, however, resistance inevitably develops over time. In order to investigate mechanisms of resistance to first‐line osimertinib, we modeled acquired resistance to this drug in transgenic mouse models of EGFRL858R‐induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR – either C797S or L718V/Q (Figure 1A and 1B). Analysis of circulating free DNA data from patients with EGFR mutant lung cancer revealed that L718Q/V mutations almost always arise in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib‐resistant C797S‐containing tumors, whereas only afatinib was effective in L718Q mutant tumors (Figure 1C and 1D). Combination first‐line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. Our data identify specific secondary EGFR mutations as a major mechanism of acquired resistance to first‐line osimertinib treatment and highlight potential strategies to overcome or prevent osimertinib resistance in vivo. Furthermore, these findings emphasize how knowledge of the specific characteristics of resistance mutations are important for determining potential subsequent treatment approaches. Support or Funding Information This work was supported by ‐‐‐‐‐Yale’s Specialized Program of Research Excellence in Lung Cancer grant (to K. Politi, S.B. Goldberg and M.A. Lemmon) and funding from AstraZeneca (to K. Politi). Additional support came from the NIH/NCI‐funded Yale Cancer Biology Training Program T32 CA193200‐01A1 and F31 CA228268‐01A1 (to J.H. Starrett), R01 CA198164 (M.A. Lemmon), the Ginny and Kenneth Grunley Fund for Lung Cancer Research, and the Canadian Institutes of Health Research Project Grant PJT‐148725 (to W.W. Lockwood). W.W. Lockwood is supported by a Michael Smith Foundation for Health Research Scholar and NIHR New Investigator Awards, A. Guernet is a fellow funded by the IMED AstraZeneca postdoc program, A. Nagelberg is supported by a scholarship from the CIHR, and K.D. Ashtekar is an Arnold and Mabel Beckman Foundation Postdoctoral Fellow. Yale Cancer Center Shared Resources used for this work were in part supported by NIH/NCI Cancer Center Support Grant P30 CA016359. Acquired resistance to first‐line osimertinib arises partially due to the emergence of secondary mutations in EGFR, which are differentially sensitive to other EGFR TKIs. A. Schema of the experiment. CCSP‐rtTA;TetO‐EGFRL858R mice were administered doxycycline (dox) for the duration of the experiment and developed tumors after ~6 weeks on dox. When tumors were detected by MRI (see pre‐treatment image), osimertinib treatment was initiated (25 mg/kg QD M‐F) which elicited a response (see representative response MRI) and treated until the emergence of resistant tumors by MRI. Coronal MR images are shown...

Macrophages are a highly plastic population of innate immunity with diverse functions in homeostasis. Despite their relative abundance in tumor microenvironment (TME), the exact function of tumor-associated macrophages (TAMs) and how they are regulated remain largely unknown. Macrophages also play key roles in lipid metabolism, disruption of which can result in pathologies such as atherosclerosis. Interestingly, TME has also been characterized as a lipid-rich context, we are interested in understanding how lipids in TME affect TAMs’ pro- or anti-tumor function. Here, we firstly characterized the lipid metabolic phenotype of TAMs in a mouse melanoma model. We found that up-regulation of lipid storage and uptake is associated with the most tolerogenic TAM subset marked by high level of F4/80. We also found high expression of scavenger receptor CD36 on F4/80hi TAMs which mediates uptake of oxidized LDL (oxLDL) from TME. Using germ line knockout (Cd36−/−) and myeloid-specific deficiency (Cd36 flox/flox x Csf1r-Cre) mouse models, we observed that uptake of oxLDL by TAMs was significantly blocked, along with rescued immunosuppressive phenotype characterized by decreased expression of PD-L1, CD206 and elevated secretion of TNFa, which together contribute to slower tumor growth. We further conducted single-cell RNA sequencing on Cd36−/− and wild-type TAMs, and found several pathways as potential mechanisms for CD36-mediated pro-tumor phenotype. In conclusion, we discovered that high expression of CD36 on TAMs mediates uptake of oxLDL and contribute to pro-tumor function. Our data suggests that metabolites in TME may play crucial roles in metabolic and functional reprogramming of tumor-infiltrating immune populations.

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib are effective for treating patients with EGFR mutant lung cancer; however, drug resistance inevitably emerges. Approaches to combine immunotherapies and targeted therapies to overcome or delay drug resistance have been hindered by limited knowledge of the effect of erlotinib on tumor-infiltrating immune cells. Using mouse models, we studied the immunological profile of mutant EGFR-driven lung tumors before and after erlotinib treatment. We found that erlotinib triggered the recruitment of inflammatory T cells into the lungs. Interestingly, this phenotype could be recapitulated by tumor regression mediated by deprivation of the EGFR oncogene indicating that tumor regression alone was sufficient for these immunostimulatory effects. Erlotinib treatment also led to increased maturation of myeloid cells and an increase in CD40+ dendritic cells. Our findings lay the foundation for understanding the effects of TKIs on the tumor microenvironment and highlights potential avenues for investigation of targeted and immuno-therapy combination strategies to treat EGFR mutant lung cancer.peer-reviewed)

mechanism that emerges. Using RNA-seq data, we searched for epigenetic regulators that might be mediating the differentially expressed genes in the resistant cells. This analysis revealed that the chromatin remodeling protein SMARCA4/BRG1 is required for maintenance of the resistant phenotype in one of the models as knockdown of BRG1 sensitized cells to osimertinib. Further analysis revealed that SMARCA4 is stabilized in TKI-resistant cells, thus leading to TKI resistance. Finally, immunohistochemistry (IHC) examination of a collection of TKI-resistant patient-derived xenografts (PDXs) revealed higher levels of SMARCA4 expression in TKI-resistant tumors without on-target EGFR-dependent resistant mechanisms. To further elucidate the role of SMARCA4, we are currently performing ATAC-seq experiments that will offer insights into chromatin accessibility mediated by the protein in the resistant cells. In addition, we are assessing the protein levels of SMARCA4 in clinical specimens obtained before treatment and at the time of resistance by IHC. As new and better targeted therapies are developed, complex resistance mechanisms that involve epigenetic changes in tumors are likely to be increasingly observed. Our studies offer insights into the mechanisms that underlie such resistance that could lead to new therapeutic possibilities for these tumors.

<p>Cell-free DNA Mutation Dataset</p>