Miller, Erin

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 Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive malignancy with poor outcomes. To investigate novel therapeutic strategies, we characterized three new metastatic prostate cancer patient derived-tumor xenograft (PDTX) models and developed 3D spheroids from each to investigate molecular targeted therapy combinations including CDK4/6 inhibitors (CDK4/6i) with AKT inhibitors (ATKi). Metastatic prostate cancer tissue was collected and three PDTX models were established and characterized using whole-exome sequencing. PDTX 3D spheroids were developed from these three PDTXs to show resistance patterns and test novel molecular-targeted therapies. CDK4/6i's were combined with AKTi's to assess synergistic antitumor response to prove our hypothesis that blockade of AKT overcomes drug resistance to CDK4/6i. This combination was evaluated in PDTX three-dimensional (3D) spheroids and in vivo experiments with responses measured by tumor volumes, PSA, and Ga-68 PSMA-11 PET-CT imaging. We demonstrated CDK4/6i's with AKTi's possess synergistic antitumor activity in three mCRPC PDTX models. These models have multiple unique pathogenic and deleterious genomic alterations with resistance to single-agent CDK4/6i's. Despite this, combination therapy with AKTi's was able to overcome resistance mechanisms. The IHC and Western blot analysis confirmed on target effects, whereas tumor volume, serum PSA ELISA, and radionuclide imaging demonstrated response to therapy with statistically significant SUV differences seen with Ga-68 PSMA-11 PET-CT. These preclinical data demonstrating antitumor synergy by overcoming single-agent CDK 4/6i as well as AKTi drug resistance provide the rational for a clinical trial combining a CDK4/6i with an AKTi in patients with mCRPC whose tumor expresses wild-type retinoblastoma 1.

Abstract A novel genetically engineered mouse (GEM) model for pediatric kidney cancer expressing proximal tubule specific ASPSCR1 :TFE3 fusion gene has been developed. The Rosa26 knock-in ASPSCR1 :TFE3 mouse was crossed with SGLT2-Cre mouse that expresses Cre recombinase in the proximal tubules of the kidney. The ASPSCR1 :TFE3 fusion gene retains the DNA binding and activation domain of TFE3, whereas the N-terminal region of TFE3 is replaced with ASPSCR1 sequences. ASPSCR1 :TFE3 is known to up-regulate the Met receptor tyrosine kinase gene and induce oncogenic phenotypes such as uncontrolled cell proliferation, invasion, and metastasis. However, the molecular roles of ASPSCR1 :TFE3 are poorly understood and no standard of care (SOC) exists for pediatric TFE3 fusion renal cell carcinoma (RCC). Ultrasound (US) and histological analyses of the kidneys of 5, 6 and 7 month old mice revealed bilateral cysts and tumors, while 6-week-old mouse kidney appears normal. At gross inspection, 5-month-old mouse had cysts with a diameter of 1-2 mm while 7-month-old mouse had a polycystic kidney and in the end stage disease. Histopathology revealed neoplastic changes in initial stages in the 5-month-old mouse whereas the neoplasia was predominantly a papillary cystic renal carcinoma; solid tumor formations were observed in the 7-month-old mouse. Neoplastic cells seen in within the blood vessel walls of the 7-month-old mouse indicated future metastasis. This new TFE3 fusion GEM will provide an avenue to understand molecular events of tumorigenesis and progression as well as a model for testing novel therapeutics with survival benefit for a cancer with no current SOC. Citation Format: Aylin Alasonyalilar-Demirer, James L. Miller, Erin E. Miller, Justyna J. Gleba, Han W. Tun, John A. Copland. ASPSCR1-TFE3 gene fusion in transgenic mouse as a new pediatric kidney cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3041.

Abstract Cancer cells increase lipid metabolism by up-regulation lipogenic enzymes such as stearoyl-CoA desaturase 1 (SCD1). It is the rate-limiting enzyme in the de novo synthesis of fatty acids (FAs), catalyzing the conversion and biosynthesis of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). We developed SSI-4, a novel highly specific SCD1 inhibitor. The anti-tumor activity ofSSI-4 was examined against different cholangiocarcinoma (CCA) models that included human and mouseCCA cell lines, CCA patient-derived xenografts (PDX) NOD SCID gamma (NSG) mouse models, and CCA PDX derived organoids. Using cell proliferation assay, we examined twelve CCA cell lines and 4 were very sensitive to SSI-4 (1-40 nM IC50). The remaining CCA cell lines showed moderate to no sensitivity to SSI-4. Studies at the protein level have shown that SCD1 is present in both sensitive and non-sensitive cells. Thus, SCD1 must be present in sensitive cells but is not predictive of response to an SCD1 inhibitor. In an effort to identify biomarkers predicting response to therapy, we performed assays, such as lipid, lactate, or energy phenotype which identified elevated lactate and unsaturated fatty acid levels as well as a glycolytic energy phenotype. In vivo, CCA PDX models have demonstrated that SSI-4strongly inhibited tumor growth. Immunohistochemical analysis of CCA tumors revealed elevated glycolytic genes in sensitive cells and thus, a potential predictive biomarker. We are currently pursuing a better understanding of the relationship between glycolysis and fatty acid synthesis/metabolism. We are also testing novel combination therapy of SSI-4 with the standard of care and other drugs to develop novel treatments for CCA. Using CCA PDX derived organoids showed synergistic antitumor activity of SSI-4in combination with cancer standard of care drugs such as gemcitabine and cisplatin. Collectively, we expect to develop a biomarker-driven selection of CCA patients sensitive to SCD1 inhibitors and novel SSI-4 combination therapy leading to improved patient survival. Citation Format: Justyna J. Gleba, Laura A. Marlow, Erin E. Miller, James L. Miller, Aylin Alasonyalilar-Demirer, Yi Guo, Kabir Mody, Lewis R. Roberts, Steven R. Alberts, Mark J. Truty, Tushar C. Patel, John A. Copland. Defining stearoyl-CoA desaturase 1 as a molecular therapeutic target against cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1004.

During branching morphogenesis, a simple cluster of cells proliferates and branches to generate an arborized network that facilitates fluid flow. The overall architecture of the mouse lung is established by domain branching, wherein new branches form laterally off the side of an existing branch. The airway epithelium develops concomitantly with a layer of smooth muscle that is derived from the embryonic mesenchyme. Here, we examined the role of smooth muscle differentiation in shaping emerging domain branches. We found that the position and morphology of domain branches are highly stereotyped, as is the pattern of smooth muscle that differentiates around the base of each branch. Perturbing the pattern of smooth muscle differentiation genetically or pharmacologically causes abnormal domain branching. Loss of smooth muscle results in ectopic branching and decreases branch stereotypy. Increased smooth muscle suppresses branch initiation and extension. Computational modeling revealed that epithelial proliferation is insufficient to generate domain branches and that smooth muscle wrapping is required to shape the epithelium into a branch. Our work sheds light on the physical mechanisms of branching morphogenesis in the mouse lung.

Mechanical forces are increasingly recognized to regulate morphogenesis, but how this is accomplished in the context of the multiple tissue types present within a developing organ remains unclear. Here we use bioengineered “microfluidic chest cavities” to precisely control the mechanical environment of the fetal lung. We show that transmural pressure controls airway branching morphogenesis, the frequency of airway smooth muscle contraction, and the rate of developmental maturation of the lungs, as assessed by transcriptional analyses. Timelapse imaging reveals that branching events are synchronized across distant locations within the lung, and are preceded by long-duration waves of airway smooth muscle contraction. Higher transmural pressure decreases the interval between systemic smooth muscle contractions and increases the rate of morphogenesis of the airway epithelium. These data reveal that the mechanical properties of the microenvironment instruct crosstalk between different tissues to control the development of the embryonic lung.

Abstract Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). Experimental Design: A breast tissue matched case–control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm2 for CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages and quantification of positive pixel measure for CD11c (dendritic cells). Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8+ T cells, CD11c+ dendritic cells, CD20+ B cells, and CD68+ macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20+ cell density (P = 0.04). Nearly 42% of BBD cases had no CD20+ B cells in evaluated lobules compared with 28% of BBD controls (P = 0.02). The absence of CD20+ cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4–23.1) for subsequent breast cancer risk. Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945–52. ©2017 AACR.

Abstract Basal subtype breast cancers have a particularly poor prognosis, with high invasiveness and resistance to most targeted therapies. TGFβ and MYC drive central features of basal breast cancer: TGFβ is an autocrine and paracrine signaling factor that drives cell invasion and metastasis, and MYC is a central regulator of cellular proliferation that is upregulated in many cancer types. We show here that genetic or pharmacologic inhibition of MYC in MCF10A basal breast cells results in increased sensitivity to TGFβ-stimulated invasion and metastasis and also show that this signaling loop is dependent on activation of SRC. Analysis of human breast cancer datasets and additional experiments with breast cancer cell lines further suggest the relevance of this signaling loop in basal, but not luminal, breast cancers. Our results imply precaution should be taken when utilizing therapeutic inhibitors of MYC with basal breast cancer patients as this could lead to increased metastasis; however, simultaneous pharmacologic inhibition of SRC and MYC for these patients could facilitate the antiproliferative effects of MYC inhibition while blocking the consequent promotion of metastasis. Cancer Res; 76(12); 3520–30. ©2016 AACR.