Cx25 contributes to leukemia cell communication and chemosensitivity

Abstract

Leukemia encompasses several hematological malignancies with shared phenotypes that include rapid proliferation, abnormal leukocyte self-renewal, and subsequent disruption of normal hematopoiesis. While communication between leukemia cells and the surrounding stroma supports tumor survival and expansion, the mechanisms underlying direct leukemia cell-cell communication and its contribution to tumor growth are undefined. Gap junctions are specialized intercellular connections composed of connexin proteins that allow free diffusion of small molecules and ions directly between the cytoplasm of adjacent cells. To characterize homotypic leukemia cell communication, we employed in vitro models for both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and measured gap junction function through dye transfer assays. Additionally, clinically relevant gap junction inhibitors, carbenoxolone (CBX) and 1-octanol, were utilized to uncouple the communicative capability of leukemia cells. Furthermore, a qRT-PCR screen revealed several connexins with higher expression in leukemia cells compared with normal hematopoietic stem cells. Cx25 was identified as a promising adjuvant therapeutic target, and Cx25 but not Cx43 reduction via RNA interference reduced intercellular communication and sensitized cells to chemotherapy. Taken together, our data demonstrate the presence of homotypic communication in leukemia through a Cx25-dependent gap junction mechanism that can be exploited for the development of anti-leukemia therapies.

Keywords: Connexin 25; cell-cell communication; gap junctions; leukemia.

Figure 1. Functional gap junction activity is detectable directly between leukemia cells

A. Jurkat cells were labeled with Calcein AM or DiI and then co-incubated for 1-3 hr. After incubation, cells were analyzed by flow cytometry. B. Quantification of the percent of dye transfer between leukemia cells as a function of time (1, 2, and 3 hr). C. Histogram of Calcein intensity in DiI-labeled Jurkat cells over time. D. Histogram of Calcein intensity in primary patient-derived AML cells, THP1 and MV4-11.

Figure 2. Pharmacological blockade of gap junctions is sufficient to attenuate communication between JURKAT and MV4-11 cells

Gap junction activity was inhibited by two pan-gap junction inhibitors, CBX and 1-octanol, at pharmacologically relevant concentrations. After 1 hr of incubation, dye transfer was reduced in cells treated with the inhibitors.

Figure 3. qRT-PCR analysis of connexin expression in leukemia

mRNA profiles of connexin expression were interrogated in normal HSCs, Jurkat cells, two primary patient-derived AML cell specimens, and two AML cell lines, MV4-11 and THP1. Two connexins were found to be more highly expressed in all leukemia cells versus normal HSCs, Cx25 and Cx40, while primary AML cell lines expressed higher levels of Cx31.9 compared with HSCs.

Figure 4. Targeting Cx25 by RNAi decreases cell-cell communication

A. Immunoblot analysis of Jurkat cells, two primary patient-derived AML cell lines, and two AML cell lines to assess Cx25 protein expression. B. Inhibition of Cx25 by shRNA-mediated knockdown showed decreased protein expression following transduction with two targeting constructs. qRT-PCR was utilized to validate the shRNA constructs and determined that both KD13 and KD36 were able to decrease Cx25 mRNA expression. C. Following inhibition of Cx25, Calcein dye transfer was reduced in Jurkat cells at 1 hr compared with the NT control.

Figure 5. Cx25 KD increases leukemia cell chemosensitivity, while treatment with a gap junction inhibitor decreases leukemia cell proliferation

A. Treatment of Cx25-knockdown Jurkat and MV4-11 cells with 15 nM Ara-C reduced their proliferative activity. B. Inhibiting gap junction activity in Jurkat cells, one primary patient-derived AML specimen, and two AML cell lines with 100 μM CBX reduced cell proliferation.

Figure 6. Cx25 expression is elevated in leukemia cell lines compared with additional tumor cell lines and connexin subunits

Box and whisker plots of connexin subunit expression across multiple cancer cell lines demonstrates that Cx25 is significantly elevated in leukemia cell lines (black box) compared with other tumor types. Data accessed from the Beroukhim et al., Nature 2010 dataset [21] in Oncomine and number of tumor cell lines per groups is indicated for each tumor. Boxes span 25th-75^th percentile, line represents median value, bars represent expression range, and *** p < 0.001.