Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world. It is characterized by the clonal expansion of CD5+CD23+ B cells in blood, marrow, and second lymphoid tissues. Survival of CLL cells are supported by cells within the tissue microenvironment and by signals from the extracellular matrix, which in part are mediated via interactions with CD44, a surface glycoprotein that is expressed by CLL B cells and by putative cancer stem cells. CD44 interacts with other surface and cytosolic proteins expressed by CLL cells such as CD38, CD49d, MMP9 and ZAP-70, which enhanced signaling leading to growth or survival of CLL cells. We found that a mAb specific for CD44 was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. It could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspase-dependent apoptosis (Proc Natl Acad Sci, USA 2013, PMID: 23530247). The cytotoxic effect of this mAb was not mitigated when the CLL cells were co-cultured with mesenchymal stromal cells (MSCs), suggesting that targeting CD44 may provide an effective approach for anti-cancer therapy.
A6 is an 8-amino acid peptide (Ac-KPSSPPEE-Am) that binds to the CD44 receptor within a region of the ligand-binding domain and induces anti-metastatic and anti-migration activity against a variety of cancers including ovarian, lung, and breast cancers. In this study we examined the cytotoxic effects and explored mechanism of A6 peptide against multiple CLL patient samples.
We found that A6 peptide exhibits a significant cytotoxic effect against CLL patient samples (N=22) in vitro while having negligible toxicity against healthy patient B cell samples (N=6). A6 also exhibits cytotoxic effects against CLL cells in a dose dependent manner. More importantly, we found that A6 is able to down-modulate CD44 expression, leading to down regulation of CD44-associated proteins (e.g. ZAP-70). Because ZAP-70 can directly enhance BCR signaling, reduction of ZAP-70, following A6 treatment resulted in attenuation of intracellular [Ca2++] flux after IgM ligation, which is an indicator of decrease in BCR signaling. Moreover, A6 exhibits significantly greater direct cytotoxicity for CLL cells (p=0.029) that express ZAP-70 than for CLL cells that were ZAP-70 negative. We transplanted primary ZAP-70 positive CLL patient samples (N=2) into the peritoneal cavity of immunodeficient mice. Intraperitoneal treatment with 300 mg/kg of A6 peptide twice daily for 7 days resulted in a significant reduction of CLL burden (90% and 64.2%) compared to PBS treated control groups. No significant adverse effects were observed. A6 peptide has been very well-tolerated in clinical trials with no clearly identifed systemic adverse events in patients with solid tumors who have been treated to date (N = 40). Thus, A6 presents a promising therapeutic agent not only against CLL but also against CD44 expressing cancer stem cells.
Citation Format: Hsien Lai, Suping Zhang, Christina Wu, Liguang Chen, Grace Liu, RongRong Wu, Fitzgerlad Lao, Jian Yu, Laura Rassenti, Michael Choi, Stephen Howell, Malcolm Finlayson, Thomas Kipps. Selective cytotoxicity of A6 peptide against ZAP-70 expressing CLL B-cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2014-950