β-catenin and PI3Kδ inhibition expands precursor Th17 cells with heightened stemness and antitumor activity

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Abstract

ICOS costimulation generates Th17 cells with durable memory responses to tumor. Herein, we found that ICOS induces PI3K/p110δ/Akt and Wnt/β-catenin pathways in Th17 cells. Coinhibiting PI3Kδ and β-catenin altered the biological fate of Th17 cells. Th17 cells inhibited of both pathways expressed less RORγt, which, in turn, reduced their ability to secrete IL-17. Unexpectedly, these cells were more effective (than uninhibited cells) at regressing tumor when infused into mice, leading to long-term curative responses. PI3Kδ inhibition expanded precursor Th17 cells with a central memory phenotype that expressed nominal regulatory properties (low FoxP3), while β-catenin inhibition enhanced Th17 multifunctionality in vivo. Remarkably, upon TCR restimulation, RORγt and IL-17 rebounded in Th17 cells treated with PI3Kδ and β-catenin inhibitors. Moreover, these cells regained β-catenin, Tcf7, and Akt expression, licensing them to secrete heightened IL-2, persist, and eradicate solid tumors without help from endogenous NK and CD8 T cells. This finding shines a light on ways to repurpose FDA-approved drugs to augment T cell–based cancer immunotherapies.

Authors

Kinga Majchrzak, Michelle H. Nelson, Jacob S. Bowers, Stefanie R. Bailey, Megan M. Wyatt, John M. Wrangle, Mark P. Rubinstein, Juan C. Varela, Zihai Li, Richard A. Himes, Sherine S.L. Chan, Chrystal M. Paulos

First-in-human trial of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab in advanced cancer patients

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Abstract

BACKGROUND. The combination of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab overcomes intrinsic and acquired resistance in both EGFR-sensitive and EGFR-resistant preclinical models of colorectal cancer (CRC).

METHODS. Utilizing a standard 3+3 design, a phase I study was designed to determine safety, maximum tolerated dose (MTD), and dose-limiting toxicities (DLTs) of the regorafenib plus cetuximab combination among patients with advanced cancer including CRC. Comprehensive genomic profiling was performed on the exceptional responder.

RESULTS. Among the 27 patients enrolled the median age was 54 years. None of 19 patients treated at dose level 1 (cetuximab i.v. 200 mg/m² followed by 150 mg/m² weekly + regorafenib 80 mg daily) experienced a DLT, and 2 of 5 patients treated at dose level 2 (cetuximab i.v. 200 mg/m² followed by 150 mg/m² weekly + regorafenib 120 mg daily) experienced a DLT (grade 3 thrombocytopenia [n = 1] and grade 3 intra-abdominal bleed [n = 1]). Most common adverse events were grade 1 or 2 rash (20 patients). Of 24 evaluable patients, 11 (46%) patients had clinical benefit (stable disease > 6 cycles or partial response [PR]) (CRC n = 8, one patient each with head and neck cancer, carcinoma of unknown primary, and glioblastoma). A CRC patient, who progressed on anti-EGFR and regorafenib, achieved a PR (46% decrease per RECIST v1.1) lasting 15 months. Genomic profiling of an exceptional responder with response for over 27 cycles revealed hypermutated genotype with microsatellite instability (MSI).

CONCLUSION. Regorafenib 80 mg daily plus cetuximab 200 mg/m² loading dose, followed by 150 mg/m² every week is the MTD/recommended phase II dose. The combination demonstrated early signals of activity in wild-type CRC, including 1 exceptional responder with MSI high.

TRIAL REGISTRATION. clinicaltrials.gov NCT02095054

FUNDING. The University of Texas MD Anderson Cancer Center is supported by the NIH Cancer Center Support Grant CA016672. This work was supported in part by the Cancer Prevention Research Institute of Texas grant RP110584 and National Center for Advancing Translational Sciences grant UL1 TR000371 (Center for Clinical and Translational Sciences).

Authors

Vivek Subbiah, Muhammad Rizwan Khawaja, David S. Hong, Behrang Amini, Jiang Yungfang, Hui Liu, Adrienne Johnson, Alexa B. Schrock, Siraj M. Ali, James X. Sun, David Fabrizio, Sarina Piha-Paul, Siqing Fu, Apostolia M. Tsimberidou, Aung Naing, Filip Janku, Daniel D. Karp, Michael Overman, Cathy Eng, Scott Kopetz, Funda Meric-Bernstam, Gerald S. Falchook

Physiologically activated mammary fibroblasts promote postpartum mammary cancer

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Abstract

Women diagnosed with breast cancer within 5 years of childbirth have poorer prognosis than nulliparous or pregnant women. Weaning-induced breast involution is implicated, as the collagen-rich, immunosuppressive microenvironment of the involuting mammary gland is tumor promotional in mice. To investigate the role of mammary fibroblasts, isolated mammary PDGFRα⁺ cells from nulliparous and postweaning mice were assessed for activation phenotype and protumorigenic function. Fibroblast activation during involution was evident by increased expression of fibrillar collagens, lysyl oxidase, Tgfb1, and Cxcl12 genes. The ability of mammary tumors to grow in an isogenic, orthotopic transplant model was increased when tumor cells were coinjected with involution-derived compared with nulliparous-derived mammary fibroblasts. Mammary tumors in the involution-fibroblast group had increased Ly6C⁺ monocytes at the tumor border, and decreased CD8⁺ T cell infiltration and tumor cell death. Ibuprofen treatment suppressed involution-fibroblast activation and tumor promotional capacity, concurrent with decreases in tumor Ly6C⁺ monocytes, and increases in intratumoral CD8⁺ T cell infiltration, granzyme levels, and tumor cell death. In total, our data identify a COX/prostaglandin E2 (PGE2)–dependent activated mammary fibroblast within the involuting mammary gland that displays protumorigenic, immunosuppressive activity, identifying fibroblasts as potential targets for the prevention and treatment of postpartum breast cancer.

Authors

Qiuchen Guo, Jessica Minnier, Julja Burchard, Kami Chiotti, Paul Spellman, Pepper Schedin

Panobinostat acts synergistically with ibrutinib in diffuse large B cell lymphoma cells with MyD88 L265 mutations

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Abstract

Diffuse large B cell lymphoma (DLBCL) frequently harbors genetic alterations that activate the B cell receptor (BCR) and TLR pathways, which converge to activate NF-κB. While selective inhibition of BTK with ibrutinib causes clinical responses in relapsed DLBCL patients, most responses are partial and of a short duration. Here, we demonstrated that MyD88 silencing enhanced ibrutinib efficacy in DLBCL cells harboring MyD88 L265P mutations. Chemical downregulation of MyD88 expression with HDAC inhibitors also synergized with ibrutinib. We demonstrate that HDAC inhibitor regulation of MyD88 expression is mediated by STAT3. In turn, STAT3 silencing caused a decrease in MyD88 mRNA and protein levels, and enhanced the ibrutinib antilymphoma effect in MyD88 mutant DLBCL cells. Induced mutations in the STAT3 binding site in the MyD88 promotor region was associated with a decrease in MyD88 transcriptional activity. We also demonstrate that treatment with the HDAC inhibitor panobinostat decreased phosphorylated STAT3 binding to the MyD88 promotor. Accordingly, combined treatment with panobinostat and ibrutinib resulted in enhanced inhibition of NF-κB activity and caused regression of DLBCL xenografts. Our data provide a mechanistic rationale for combining HDAC inhibitors and ibrutinib for the treatment of DLBCL.

Authors

Patrizia Mondello, Elliott J. Brea, Elisa De Stanchina, Eneda Toska, Aaron Y. Chang, Myles Fennell, Venkatraman Seshan, Ralph Garippa, David A. Scheinberg, José Baselga, Hans-Guido Wendel, Anas Younes

Priming is key to effective incorporation of image-guided thermal ablation into immunotherapy protocols

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Abstract

Focal therapies play an important role in the treatment of cancers where palliation is desired, local control is needed, or surgical resection is not feasible. Pairing immunotherapy with such focal treatments is particularly attractive; however, there is emerging evidence that focal therapy can have a positive or negative impact on the efficacy of immunotherapy. Thermal ablation is an appealing modality to pair with such protocols, as tumors can be rapidly debulked (cell death occurring within minutes to hours), tumor antigens can be released locally, and treatment can be conducted and repeated without the concerns of radiation-based therapies. In a syngeneic model of epithelial cancer, we found that 7 days of immunotherapy (TLR9 agonist and checkpoint blockade), prior to thermal ablation, reduced macrophages and myeloid-derived suppressor cells and enhanced IFN-γ–producing CD8⁺ T cells, the M1 macrophage fraction, and PD-L1 expression on CD45⁺ cells. Continued treatment with immunotherapy alone or with immunotherapy combined with ablation (primed ablation) then resulted in a complete response in 80% of treated mice at day 90, and primed ablation expanded CD8⁺ T cells as compared with all control groups. When the tumor burden was increased by implantation of 3 orthotopic tumors, successive primed ablation of 2 discrete lesions resulted in survival of 60% of treated mice as compared with 25% of mice treated with immunotherapy alone. Alternatively, when immunotherapy was begun immediately after thermal ablation, the abscopal effect was diminished and none of the mice within the cohort exhibited a complete response. In summary, we found that immunotherapy begun before ablation can be curative and can enhance efficacy in the presence of a high tumor burden. Two mechanisms have potential to impact the efficacy of immunotherapy when begun immediately after thermal ablation: mechanical changes in the tumor microenvironment and inflammatory-mediated changes in immune phenotype.

Authors

Matthew T. Silvestrini, Elizabeth S. Ingham, Lisa M. Mahakian, Azadeh Kheirolomoom, Yu Liu, Brett Z. Fite, Sarah M. Tam, Samantha T. Tucci, Katherine D. Watson, Andrew W. Wong, Arta M. Monjazeb, Neil E. Hubbard, William J. Murphy, Alexander D. Borowsky, Katherine W. Ferrara

Genomic profiling reveals mutational landscape in parathyroid carcinomas

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Abstract

Parathyroid carcinoma (PC) is an extremely rare malignancy lacking effective therapeutic intervention. We generated and analyzed whole-exome sequencing data from 17 patients to identify somatic and germline genetic alterations. A panel of selected genes was sequenced in a 7-tumor expansion cohort. We show that 47% (8 of 17) of the tumors harbor somatic mutations in the CDC73 tumor suppressor, with germline inactivating variants in 4 of the 8 patients. The PI3K/AKT/mTOR pathway was altered in 21% of the 24 cases, revealing a major oncogenic pathway in PC. We observed CCND1 amplification in 29% of the 17 patients, and a previously unreported recurrent mutation in putative kinase ADCK1. We identified the first sporadic PCs with somatic mutations in the Wnt canonical pathway, complementing previously described epigenetic mechanisms mediating Wnt activation. This is the largest genomic sequencing study of PC, and represents major progress toward a full molecular characterization of this rare malignancy to inform improved and individualized treatments.

Authors

Chetanya Pandya, Andrew V. Uzilov, Justin Bellizzi, Chun Yee Lau, Aye S. Moe, Maya Strahl, Wissam Hamou, Leah C. Newman, Marc Y. Fink, Yevgeniy Antipin, Willie Yu, Mark Stevenson, Branca M. Cavaco, Bin T. Teh, Rajesh V. Thakker, Hans Morreau, Eric E. Schadt, Robert Sebra, Shuyu D. Li, Andrew Arnold, Rong Chen

Randomized, placebo-controlled window trial of EGFR, Src, or combined blockade in head and neck cancer

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Abstract

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition.

METHODS. Patients with operable stage II–IVa HNSCC were randomized to 7–21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size.

RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms (P = 0.0014); however, no effect was seen with dasatinib alone (P = 0.24). High baseline pMAPK expression was associated with response to erlotinib (P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib (P = 0.099).

CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts.

TRIAL REGISTRATION. NCT00779389.

FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma.

Authors

Julie E. Bauman, Umamaheswar Duvvuri, William E. Gooding, Tanya J. Rath, Neil D. Gross, John Song, Antonio Jimeno, Wendell G. Yarbrough, Faye M. Johnson, Lin Wang, Simion Chiosea, Malabika Sen, Jason Kass, Jonas T. Johnson, Robert L. Ferris, Seungwon Kim, Fred R. Hirsch, Kimberly Ellison, John T. Flaherty, Gordon B. Mills, Jennifer R. Grandis

RCAN1-4 is a thyroid cancer growth and metastasis suppressor

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Abstract

Metastasis suppressors are key regulators of tumor growth, invasion, and metastases. Loss of metastasis suppressors has been associated with aggressive tumor behaviors and metastatic progression. We previously showed that regulator of calcineurin 1, isoform 4 (RCAN1-4) was upregulated by the KiSS1 metastatic suppression pathway and could inhibit cell motility when overexpressed in cancer cells. To test the effects of endogenous RCAN1-4 loss on thyroid cancer in vivo, we developed RCAN1-4 knockdown stable cells. Subcutaneous xenograft models demonstrated that RCAN1-4 knockdown promotes tumor growth. Intravenous metastasis models demonstrated that RCAN1-4 loss promotes tumor metastases to the lungs and their subsequent growth. Finally, stable induction of RCAN1-4 expression reduced thyroid cancer cell growth and invasion. Microarray analysis predicted that nuclear factor, erythroid 2-like 3 (NFE2L3) was a pivotal downstream effector of RCAN1-4. NFE2L3 overexpression was shown to be necessary for RCAN1-4–mediated enhanced growth and invasiveness and NEF2L3 overexpression independently increased cell invasion. In human samples, NFE2L3 was overexpressed in TCGA thyroid cancer samples versus normal tissues and NFE2L3 overexpression was demonstrated in distant metastasis samples from thyroid cancer patients. In conclusion, we provide the first evidence to our knowledge that RCAN1-4 is a growth and metastasis suppressor in vivo and that it functions in part through NFE2L3.

Authors

Chaojie Wang, Motoyasu Saji, Steven E. Justiniano, Adlina Mohd Yusof, Xiaoli Zhang, Lianbo Yu, Soledad Fernández, Paul Wakely Jr., Krista La Perle, Hiroshi Nakanishi, Neal Pohlman, Matthew D. Ringel

Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

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Abstract

Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KL_Lenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KL_Lenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

Authors

Melissa Gilbert-Ross, Jessica Konen, Junghui Koo, John Shupe, Brian S. Robinson, Walter Guy Wiles IV, Chunzi Huang, W. David Martin, Madhusmita Behera, Geoffrey H. Smith, Charles E. Hill, Michael R. Rossi, Gabriel L. Sica, Manali Rupji, Zhengjia Chen, Jeanne Kowalski, Andrea L. Kasinski, Suresh S. Ramalingam, Haian Fu, Fadlo R. Khuri, Wei Zhou, Adam I. Marcus

Th17 cells are refractory to senescence and retain robust antitumor activity after long-term ex vivo expansion

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Abstract

Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8⁺ T cells for treatment, they also cause decline in the cell’s therapeutic fitness. In contrast, we discovered that IL-17–producing CD4⁺ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor–positive (CAR⁺) Th17 cells also retained their ability to regress human mesothelioma, while CAR⁺ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence.

Authors

Jacob S. Bowers, Michelle H. Nelson, Kinga Majchrzak, Stefanie R. Bailey, Baerbel Rohrer, Andrew D.M. Kaiser, Carl Atkinson, Luca Gattinoni, Chrystal M. Paulos