Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Lori A. Hazlehurst

Keywords

calcium, CAM-DR, HYD1, integrins, necrosis, VLA-4

Abstract

In our previous work we demonstrated that the integrin antagonist, HYD1, induced necrotic cell death in myeloma cell lines in vitro and in vivo as a single agent. In order to further delineate biomarkers of response to HYD1 we developed an isogenic drug resistant variant named H929-60. We show that the acquisition of resistance towards HYD1 correlates with reduced expression of the cleaved α4 integrin subunit and beta 1 integrin. Moreover, we demonstrate that HYD1 interacts with α4 integrin in myeloma cells. Consistent with reduced VLA-4 expression, the resistant variant showed ablated functional binding to fibronectin, VCAM-1 and the bone marrow stroma cell line, HS-5. The reduction in binding to extracellular matrices of the resistant variant translated to sensitivity to melphalan and bortezomib induced cell death in the bone marrow stroma co-culture model of drug resistance. Moreover, CD138 positive myeloma cells were more sensitive to HYD1 induced cell death compared to the CD138 negative fraction, and potency of HYD1 induced cell death significantly correlated with α4 integrin expression. We were also able to show that reducing α4 or β1 integrin using shRNA strategies was sufficient to cause resistance in myeloma cell lines. In addition we investigated the effects of cyclized variants of HYD1 to improve potency of the agent. One such compound, named HM-27, was determined to be 30 fold more active in H929 cells when compared to HYD1. HM-27 and HYD1 were determined to have similar mechanisms of action as H929-60 cells were shown to be resistant to both compounds when compared to H929 cells. We further characterized HM-27's mechanism of action by investigating what effects HM-27 induced Ca2+ oscillations had on HM-27 induced cell death. The increases in intracellular Ca2+ seen after treatment with HM-27 were determined to occur via release from ER stores and not through influx through plasma membrane channels. Inhibiting Ca2+ release from the ER also potentiated the effects of HM-27 in MM cells. Furthermore, inhibiting Ca2+ release from the ER was also shown to block the onset of autophagy after ER treatment. Treating cells with the lysosomotropic agent, chloroquine, was shown to potentiate the activity of HM-27 in vitro and ex vivo. HM-27 was also shown to have activity in an in vivo model with combination treatment containing bortezomib and HM-27 increasing mouse survival. Collectively our data indicate that VLA-4 expression is a critical determinant of response to HYD1 induced cell death. We also showed that increases in intracellular Ca2+ seen after treatment with HM-27 had a cytoprotective effect in MM cells. Moreover, neutralizing autophagy potentiates HM-27 induced cell death in vitro and ex vivo while combining bortezomib and HM-27 increased survival in vivo. These data continue to provide rationale for further pre-clinical development of HYD1 as a novel anticancer agent.

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