Smallridge, Robert C.

Background Monensin, a Na + ionophore, can increase cytosolic Ca 2+ ([Ca 2+ ] i ) by reversing the Na + /Ca 2+ exchange mechanism. This study provided additional insights into the mechanism of this Na + ionophore-induced increase in [Ca 2+ ] i , and emphasized the critical role of phospholipase C (PLC) in amplifying Na + /Ca 2+ exchange-induced Ca 2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. Methods FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca 2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3 H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. Results In Hank's balanced salt solution with Ca 2+ (HBSS+), monensin (100 µmol/L) induced a 2.3-fold sustained Ca 2+ increase associated with IP 3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca 2+ , or replacement of Na + by choline chloride in the medium, reduced the [Ca 2+ ] i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na + channel blocker, or Na + /Ca 2+ exchange inhibitors. In HBSS without Ca 2+ (HBSS-), monensin induced a 1-fold transient [Ca 2+ ] i increase but did not increase the AA. This Ca 2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca 2+ ] i , but reduced the sustained second phase of [Ca 2+ ] i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A 2 (PLA 2 ) inhibitor blocked the monensin-induced AA release without affecting the [Ca 2+ ] i increase. Inhibition of PKC prevented 87% to 94% of the monensin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. Conclusions The results suggest that monensin initiates an increase in [Ca 2+ ] i via a Na + /Ca 2+ exchange mechanism that triggers more pronounced and sustained [Ca 2+ ] i increase via activation of PLC and Ca 2+ influx. The PLC activation, followed by sustained Ca 2+ influx and PKC activation, is a prerequisite for PLA 2 -mediated processes in monensin-challenged FRTL-5 thyroid cells. Key words: FRTL-5 thyroid cells • phospholipases • protein kinase C • MAP kinase • cell calcium

Abstract Chimeric antigen receptor T (CART) cell therapy has shown unprecedented success in the treatment of hematological malignancies. However, the immunosuppressive tumor microenvironment of both solid and liquid tumors is a major barrier for CART efficacy. Immunosuppressive myeloid cells inhibit CART efficacy. Colony-stimulating factor 1 receptor (CSF1R) promotes macrophage proliferation, survival, and anti-inflammatory response and is upregulated on myeloid cells in many cancers, making CSF1R an appealing therapeutic target. We aimed to test the interactions between CSF1R, myeloid cells, and CART response, and to study CSF1R targeting in the context of CART cell therapy. We utilized CD19-targeted CART cell therapy (CART19) in lymphoma preclinical models and thyroid-stimulating hormone receptor (TSHR)-targeting CART cell therapy (TSHR-CART) in thyroid cancer models. We first measured CSF1R expression on myeloid cells in vitro. Suppressive myeloid cells, polarized from healthy donor monocytes using an anaplastic thyroid cancer (ATC) cell line, 8505c, or mantle cell lymphoma cell line, JeKo-1, significantly upregulate CSF1R (p=0.0002) compared to media control. After polarization, myeloid cells significantly inhibit TSHR-CART and CART19 antigen-specific proliferation compared to freshly isolated monocytes (p=0.012, p<0.01, respectively) indicating that suppressive myeloid cells contribute to CART failure in multiple tumor types. We then performed single cell RNA sequencing on peripheral blood taken at baseline from patients with mantle cell lymphoma treated with CART19. We found a significant increase of CSF1R on monocyte subsets in CART19 non-responders vs responders (padj=0.0037). Next, we determined the impact of CSF1R blockade on CART cell therapy in vivo. Using lymphoma models, B6 mice were engrafted with the luciferase+ CD19+ murine lymphoma cell line TBL-12 (1x106 cells i.v. 1 day after 250 mg/kg cyclophosphamide). Tumor burden was significantly reduced after 7 days of treatment with low dose CART19 (0.5x106 i.v.) + anti-mouse CSF1R antibody (16mg/kg i.p. twice weekly) vs CART19 + IgG control (p=0.0146). We then tested the impact of CSF1R blockade in mice experiencing tumor relapse. After TBL-12 engraftment, mice were treated with anti-mouse CSF1R or IgG control antibody followed two days later with high dose CART19 (1x106 i.v.) to induce remission. Mice were re-challenged with TBL-12 14 days later. Mice treated with anti-mouse CSF1R antibody showed increased tumor control and significantly higher survival (p=0.0416). Using thyroid cancer models, first we identified high macrophage infiltration in biopsy specimens of patients with ATC compared to normal thyroid tissue and benign thyroid tumors through immunohistochemistry (IHC) of patient samples (p<0.01, p<0.0001, respectively). To test if macrophages are associated with CART failure in ATC, NSG mice were subcutaneously engrafted with 1x106 TSHR+ THJ529 tumors, an ATC cell line derived from patient biopsy samples from the Mayo Clinic thyroid cancer biobank, followed by i.v. treatment with 1x107 TSHR-CART or untransduced T cell control (UTD). IHC staining of tumor tissue collected at experimental endpoints revealed a dose-dependent, significant infiltration of human CD3+ T cells into the tumors which inversely correlated with murine F4/80+ myeloid cell and murine YM-1+ M2 macrophage infiltration. This led us to hypothesize that myeloid cell targeting with CSF1R blockade can improve TSHR-CART efficacy. We utilized the same ATC model described above to test CSF1R targeting. When TSHR+ THJ529 tumors reached approximately 100 mm3, mice were randomized by tumor volume and treated with CSF1R inhibitor PLX5622 (600mg/kg) or vehicle control, followed by either UTD or TSHR-CART cells 5x106 i.v. Mice treated with TSHR-CART + PLX5622 showed significantly improved tumor control vs TSHR-CART + vehicle control (p<0.0001) or UTD + PLX5622 (p<0.0001). Together, our results highlight immunosuppressive myeloid cells which express CSF1R as a major cell type limiting CART efficacy in both solid and liquid tumors. We also identified a strategy to overcome myeloid-mediated suppression of CART cells by targeting CSF1R with antibody or small molecule inhibitors.

Abstract Oncocytic carcinoma (Hurthle cell carcinoma) has the highest incidence of metastasis among the differentiated thyroid cancers. Metastatic disease is reported at the initial diagnosis in 10-20% of patients and 34% overall. Thyroid-stimulating hormone receptor (TSHR) expression is limited to the thyroid gland. It is abundantly expressed on thyroid tumor cells, making TSHR a compelling target for advanced thyroid cancer diagnostics and therapeutics. We developed a potent anticancer strategy for treating oncocytic carcinoma using a novel thyroid hormone stimulating receptor (TSHR)- targeted (chimeric antigen receptor) CAR T cell therapy. TSHR CAR T was generated by transducing T cells derived from healthy donors. In vitro effector functions (cytotoxicity, proliferation, degranulation) were tested in TSHR+ tumor models, and subsequent in vivo studies using NSG mice were performed to assess anti-tumor activity efficacy. We observed strong anti-tumor activity against metastatic oncocytic carcinoma cell line XCT.UC1 using the TSHR CAR T therapy in vivo. Re-challenging treated mice with XCT.UC1 cells reactivated TSHR CART cells with enhanced proliferation and complete suppression of tumor growth of the newly injected tumor cells. Our work represents a promising strategy to improve treatment outcomes of patients with metastatic oncocytic carcinoma that may provide a durable response. Citation Format: Justyna J. Gleba, Aylin Alasonyalilar-Demirer, Matthew Pawlush, Claudia Manriquez-Roman, Elizabeth L. Siegler, Robert C. Smallridge, Abba Zubair, Han W. Tun, Saad S. Kenderian, John A. Copland. Novel TSHR CAR T therapy for targeting oncocytic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5082.

Abstract Thyroid cancer is the most common endocrine cancer in the US, and its incidence is rising. Most thyroid cancer deaths are attributed to treatment-refractory, metastatic tumors. Thyroid stimulating hormone receptor (TSRH) expression is largely limited to the thyroid gland and is abundantly expressed on thyroid tumor cells, making TSRH a compelling target for advanced thyroid cancer diagnostics and therapeutics. Therefore, we developed a novel TSHR-targeted chimeric antigen receptor (CAR) T cell therapy to treat aggressive thyroid cancers. TSHR-CAR constructs were cloned into a lentiviral CAR construct containing 4-1BB and CD3ζ. First, we demonstrated potent TSHR-CART antigen-specific anti-tumor activity in vitro. Then, NOD-SCID-γ-/- (NSG) mice were inoculated subcutaneously with TSHR+ tumor cells and randomized by tumor volume to treatment with TSHR-CART cells or control Untransduced T cells (UTD). Treatment with TSHR-CART cells resulted in dose-dependent antitumor activity and prolonged survival. De-differentiated anaplastic thyroid cancers (ATC) downregulate TSHR. Our TSHR immunohistochemistry results corroborated these findings and displayed minimal TSHR protein expression, precluding successful TSHR-CART treatment. We therefore sought to sensitize these tumors with MAPK inhibitors, as a strategy to upregulate TSHR expression in patients with metastatic thyroid cancer. TSHR expression was upregulated in patient-derived xenograft (PDX) ATC models after one week of daily administration of the MAPK inhibitors (p=0.0024). After confirming that MAPK inhibition does not dampen TSHR-CART effector functions, we tested sequential and combination therapy of TSHR-CART with MEK and BRAF inhibition in vivo. NSG mice were engrafted with ATC BRAF-mutant PDX tumors and randomized by tumor volume to daily oral treatment with placebo or trametinib (MEK inhibitor) plus dabrafenib (BRAF inhibitor). One week later, mice received either UTD or TSHR-CART. Mice conditioned with trametinib plus dabrafenib (p=0.0018) and subsequently treated with TSHR-CART showed superior antitumor activity. However, the improved antitumor activity in this setting was transient. We therefore tested the durability of TSHR upregulation following MEK/BRAF inhibition and demonstrated that TSHR upregulation lasts less than 48-72 hours after discontinuation. Finally, we tested the combination of TSHR CART cells with MEK/BRAF inhibitors in ATC BRAF-mutant PDX tumors. Here, combining TSHR-CART cells with MEK/BRAF inhibitors result in durable control of the tumors. Collectively, our findings indicate that MEK/BRAF inhibition of de-differentiated thyroid cancers upregulated TSHR expression and enhanced TSHR-CART antitumor activity. This work represents a viable strategy to improve outcomes of patients with aggressive, metastatic thyroid cancers. Citation Format: Claudia Manriquez Roman, Kendall J. Schick, Justyna J. Gleba, Truc N. Huynh, Elizabeth L. Siegler, James L. Miller, Aylin Alasonyalilar Demirer, Matthew L. Pawlush, Ahmet Biligili, Long K. Mai, Erin Tapper, Leo R. Sakemura, Michelle J. Cox, Carli M. Stewart, Ismail Can, Ekene J. Ogbodo, Gaofeng Cui, Georges Mer, Gloria R. Olivier, Yushi Qiu, Robert C. Smallridge, Zubair C. Abba, Han W. Tun, John A. Copland, Saad S. Kenderian. Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5074.

Background: Anaplastic thyroid cancer (ATC) is a rare but highly lethal form of thyroid cancer. Since the guidelines for the management of ATC by the American Thyroid Association were first published in 2012, significant clinical and scientific advances have occurred in the field. The aim of these guidelines is to inform clinicians, patients, and researchers on published evidence relating to the diagnosis and management of ATC. Methods: The specific clinical questions and topics addressed in these guidelines were based on prior versions of the guidelines, stakeholder input, and input of the Task Force members (authors of the guideline). Relevant literature was reviewed, including serial PubMed searches supplemented with additional articles. The American College of Physicians Guideline Grading System was used for critical appraisal of evidence and grading strength of recommendations. Results: The guidelines include the diagnosis, initial evaluation, establishment of treatment goals, approaches to locoregional disease (surgery, radiotherapy, targeted/systemic therapy, supportive care during active therapy), approaches to advanced/metastatic disease, palliative care options, surveillance and long-term monitoring, and ethical issues, including end of life. The guidelines include 31 recommendations and 16 good practice statements. Conclusions: We have developed evidence-based recommendations to inform clinical decision-making in the management of ATC. While all care must be individualized, such recommendations provide, in our opinion, optimal care paradigms for patients with ATC.

BackgroundFactors associated with receiving initial care for thyroid cancer (TC) at academic centers (ACs) versus nonacademic centers (NACs) and their impact on patient outcomes have not been reported.MethodsThe National Cancer Database with TC cases from 2004 to 2013 was evaluated for association of type of center for initial care with socioeconomic factors and disease and treatment characteristics, as well as overall survival (OS; all‐cause mortality).ResultsThe patients with TC (n = 200,824) included were predominantly women (74%), non‐Hispanic Whites (85%), and from metro areas (84%). Sixty percent received initial care at a NAC. There were no significant differences between treatment groups by age or gender. Among those treated at an AC, a higher proportion belonged to racial/ethnic minorities (16.5%) versus at a NAC (11.6%). Hormone therapy was used more in an AC versus a NAC (60% vs 47%). Patients with all TC pathologies combined had a lower likelihood of death when they received initial care at an AC (hazard ratio [HR], 0.948; P = .0006). Among individual pathologic subtypes, a lower likelihood of death was noted when initial care was received at an AC for follicular (HR, 0.828, P = .0010) and Hurthle cell cancers (HR, 792; P = .0008), as well as stage II papillary thyroid cancer (HR, 0.828; P = .0026), but not for other histopathologic subtypes.ConclusionsInitial care at an AC was associated with lower likelihood of death for patients with TC, especially for those with follicular or Hurthle cell subtypes. Optimal resource use with consideration of patients' socioeconomic and demographic factors is imperative to ensure the most appropriate management of patients with TC in various treatment settings.