Epigenetic Modulation of Alemtuzumab Mediated Antileukemia Effects and Resistance in T- Cell Prolymphocytic Leukemia
Authors: Sharma Kamal , Shah Mithun , Hasanali Zainul , Ghai Vikas , Broeg Katie , Shimko Sara , Stuart August , Epner Elliot M , Loughran Thomas P. 
Published: October 2019

Abstract
Abstract 2440
T-Cell prolymphocytic leukemia (T-PLL) is an aggressive chemotherapy-resistant hematological malignancy. This disease is more frequently reported in men and is exclusively reported in adults. Raised lymphocyte count, skin lesions and enlarged organs are the frequent clinical symptoms evident in this disease. T-PLL affects mature post-thymic T-cells. Most cases have the CD4+CD8- phenotype; however, 25% of cases do have the CD8+/CD4- phenotype. Currently there is no treatment available that achieves durable complete remission of T-PLL. Current frontline treatment consists of alemtuzumab, an anti CD52 antibody that is expressed on T and B cells and in most PLL patients. This treatment induces complete remissions (CR) in the majority (>50%) of patients that are not durable; median duration of remission is 6 months.
The first T-PLL patient exhibited de novo alemtuzumab resistance that we hypothesized was epigenetic in nature. Based on our work and that of others in B cell malignancies, we added cladribine (2-cda; 5mg/m2 d 1–5) while continuing alemtuzumab. The patient had a dramatic decline in her WBC count (Fig. 1) and achieved a peripheral blood complete remission by flow cytometry and PCR. Patient 2 was treated with alemtuzumab alone with complete remission that lasted 6 months when he relapsed with WBC count of 600,000 (Fig. 1). He underwent pheresis and was initiated on treatment with alemtuzumab and cladribine with some response but a pattern consistent with epigenetic resistance. The histone deacetylase inhibitor (HDACi), SAHA (vorinostat), was added (400 mg daily × 5 days) with a rapid decline in WBC count and CR by flow cytometry (Fig. 1). Based on these encouraging results, we have treated 3 additional patients in the frontline setting with alemtuzumab and cladribine (Fig. 1). All have responded with rapid decline in WBC counts with complete remission by flow cytometry and PCR. All of these newly diagnosed patients remain in complete remission by flow cytometry and PCR with followup of 1–14 months.
Correlative science experiments have included transcriptional arrays and well as Q-RT PCR for candidate genes identified using the HELP (Hpa II enriched Ligation Mediated PCR)assay in mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) patients treated with cladribine and rituximab with or without SAHA. Microarray gene expression analysis was performed using total RNA from four normal donors and four T-PLL patients. One pre-treatment and one post-treatment sample was obtained and analyzed from each of these four T-PLL patients. The whole blood samples that were acquired from the normal donors were CD3 enriched and tested for purity. Data obtained was normalized and run on the Gene Set Enrichment Analysis (GSEA) program. Biological gene patterns were analyzed between the pre-treatment and post-treatment T-PLL samples. After analysis, it was apparent that the Jak-Stat gene set was significantly enriched (p-value 0.006 and FDR of 0.068) in the pre-treatment T-PLL samples when compared to the post-treatment samples. The p53 gene and p53 pathway genes were transcriptionally upregulated, but did not reach statistical significance. Q-RT PCR validation studies were conducted. One patient had a 5 fold increase in p53 mRNA and another had a 13 fold increase in DUSP2 mRNA (Fig. 2), a phosphatase that is a downstream target of p53. P53 has been not thought to be epigenetically regulated before a recent paper utilizing adenovirus in cultured cells. These results demonstrate that resistance to monoclonal antibody antitumor effects can be overcome with epigenetic agents. Correlative science data implicates the p53 gene pathway in mediating monoclonal antibody induced antileukemia effects in PLL, and imply that inhibition of Jak-Stat signaling may also play a role in these cells. We also demonstrate epigenetic regulation of p53 and a downstream target gene (DUSP2) that correlates with response to alemtuzumab. Furthermore, our data suggests that the combination of epigenetic agents with monoclonal antibodies should be studied and that epigenetic agents are potentially capable of activating multiple silenced genes in different pathways. A prospective, multicenter trial of alemtuzumab, cladribine, with or without an HDACi in newly diagnosed and relapsed PLL is warranted.

Disclosures:
Epner: Merck: Consultancy, Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Millenium: Speakers Bureau; Allos: Speakers Bureau; Enzon: Speakers Bureau; GSK: Speakers Bureau.