In-Kyung Park

Colorectal Cancer Stem Cells Are Enriched in Xenogeneic Tumors Following Chemotherapy

Background Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA+CD44+ phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. Methods and Findings Xenogeneic tumors initiated with CoCSC were allowed to reach ∼400 mm3, at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. Conclusions CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

DLL4 Blockade Inhibits Tumor Growth and Reduces Tumor-Initiating Cell Frequency

Previous studies have shown that blocking DLL4 signaling reduced tumor growth by disrupting productive angiogenesis. We developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. We found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes and reduced proliferation of tumor cells. Furthermore, we found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.

Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors

The Wnt/β-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.

A Genetic Determinant That Specifically Regulates the Frequency of Hematopoietic Stem Cells

The regulation of hematopoietic stem cell (HSC) homeostasis is not well understood. We screened for genetic polymorphisms that were linked to differences between mouse strains in the numbers of long-term reconstituting HSCs or restricted progenitors in the bone marrow. AKR/J mice had significantly higher frequencies and numbers of both HSCs and restricted progenitors in their bone marrow than C57BL/Ka-Thy-1.1 mice. The C57BL/Ka-Thy-1.1 alleles were partially dominant. A locus on chromosome 17, including the H-2 complex, was significantly linked to the frequency of long-term self-renewing HSCs but showed no evidence of linkage to the frequency of restricted progenitors. Conversely, a chromosome 1 locus exhibited suggestive linkage to restricted progenitor frequencies but was not linked to HSC frequency. This demonstrates that there are distinct genetic determinants of the frequencies of HSCs and restricted progenitors in vivo. The AKR/J chromosome 17 locus was not sufficient to increase HSC frequencies when bred onto a C57BL background. This suggests that to affect HSC frequencies, the product(s) of this locus likely depend on interactions with unlinked modifying loci.

Abstract A058: GITRL-Fc, an immunotherapeutic agent that stimulates T-cell-mediated antitumor immune response

GITRL, (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF family of ligands and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. A novel single-gene linkerless GITRL trimer was shown to be functional when fused to either the N- or C-terminus of an immunoglobulin Fc domain, offering a flexible strategy that may also be amenable to the production of bispecific agents. GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in several murine tumor graft models, including the apparent total regression of some treated tumors. GITRL-Fc potentiated tumor specific T-cell responses, particularly of the Th1 type, increased antigen-specific CD8 response, and promoted a reduction in Treg-mediated immune-suppressive activity. Citation Format: Fumiko Axelrod, Hyun-Bae Jie, Erin Mayes, Jorge Monteon, Minu Srivastava, Rui Yun, Inkyung Angie Park, Austin Gurney. GITRL-Fc, an immunotherapeutic agent that stimulates T-cell-mediated antitumor immune response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A058.