CD30 regulation by chemical and viral agents and the resulting effect on anti-CD30 therapy

Open Access
Author:
Hasanali, Zainul Shoyeb
Graduate Program:
Molecular Medicine
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 06, 2015
Committee Members:
  • Clare E Sample, Dissertation Advisor
  • Thomas Loughran Jr, Dissertation Advisor
  • Clare E Sample, Committee Chair
  • Leslie Joan Parent, Committee Member
  • Diane M Thiboutot, Committee Member
  • Neil David Christensen, Committee Member
Keywords:
  • CD30
  • brentuximab vedotin
  • EBV
  • T-PLL
  • LMP1
  • cladribine
  • vorinostat
  • alemtuzumab
  • romidepsin
  • SAHA
  • BL
Abstract:
Cancer treatment is beginning to follow the personalized medicine model. An effective example of personalized therapy is the drug brentuximab vedotin, a humanized antibody against the cell surface receptor CD30 conjugated to monomethyl auristatin E. CD30 is expressed mainly on the surface of activated lymphocytes. Brentuximab vedotin targets CD30-positive cells for auristatin E, a potent and effective mitotic spindle toxin. The side effect profile is low and efficacy high in CD30-positive cells. CD30 is a great drug target, but there are few CD30-positive leukemias and lymphomas. But what if there was a way to force CD30 expression? It would open the door for brentuximab vedotin use in other lymphoid malignancies. The induction of CD30 expression is the main focus of this dissertation. We approached this goal in two ways, to uncover agents that could induce CD30 and to understand the mechanism by which induction occurs. We performed three independent studies in pursuit of this goal. Our first study was directed at finding a better treatment for the aggressive and rapidly fatal disease T-cell prolymphocytic leukemia (T-PLL). Treatment with monoclonal antibody alemtuzumab, anti-CD52, has good efficacy in treatment naïve patients, but relapse is inevitable and refractory to further alemtuzumab treatment. We used the epigenetic drugs, cladribine, a DNA methyltransferase inhibitor, and vorinostat, a histone deacetylase inhibitor, in combination with alemtuzumab. We noted that all patients responded and remained amenable to treatment even after relapse. This treatment has the potential to slow an aggressive and fatal disease and to allow time for bone marrow transplant, the only currently curative measure. During this study, we discovered that certain patients had upregulated the expression of CD30 after epigenetic treatment. This finding was useful, because it allowed for brentuximab vedotin treatment of a patient that had alemtuzumab resistant T-PLL in his skin. Epigenetic drugs maintain treatment susceptibility and create new treatment targets such as CD30. During our T-PLL study, we noticed that patients treated with only cladribine and alemtuzumab were CD30-positive. After addition of vorinostat, CD30 expression was silenced. One patient was switched from vorinostat to romidepsin and then induced CD30. To study this phenomenon more closely, we tested the effects of vorinostat treatment of three CD30-positive cell lines in vitro. We found that vorinostat reversibly silenced CD30 mRNA and protein expression. This finding indicates that HDAC enzymes are critical to the expression of CD30. We also tested whether vorinostat mediated decreases in CD30 had an effect on the efficacy of brentuximab vedotin. All three cell lines were susceptible to brentuximab vedotin prior to the addition of vorinostat, but all three became significantly less so after concurrent treatment. If a subclinical concentration of vorinostat was used that did not decrease CD30 levels by more than 40-50%, the effect of brentuximab vedotin was increased. As long as the target for brentuximab vedotin, CD30, remained high enough, the anti-tumor effects of vorinostat were additive. Several previous studies have linked some EBV positive tumors to CD30 expression. EBV enters into latency in order to evade the immune system and persist. There are three latency states expressing unique patterns of latency proteins. Using Burkitt lymphoma cell lines that were either in Latency I or III, we showed that Latency III was CD30 positive and Latency I was not. This explained why some EBV-positive tumors are CD30-positive and others are not. By expressing latent membrane protein 1 (LMP1) in an EBV-negative cell line, we showed that it is responsible for CD30 upregulation. In order for LMP1 to upregulate CD30, the CD30 promoter has to be hypomethylated, specifically the region around the Sp1 transcription factor binding site. LMP1 triggers all arms of the MAP kinase, Akt and NF-κB pathways through its intracellular domains. This signaling activates expression of c-jun and JunB, two major regulators of the CD30 promoter, and leads to its expression. We also proved that an LMP1-negative, CD30-negative Latency I cell line could be treated with 5-azacytidine, a DNA methyltransferase inhibitor, and forced to express low levels of LMP1 and CD30. EBV positive Latency I tumors could be treated with 5-azacytidine or a similar drug to force expression of CD30 and then brentuximab vedotin to kill CD30 positive cells. These studies lay the foundation for a new treatment option in the fight against EBV positive lymphoid malignancies. Our studies provide proof of concept for CD30 induction and highlight some of the mechanisms involved. These findings lay the groundwork for the expansion of brentuximab vedotin treatment into EBV-positive lymphoid malignancies.