One major focus of the Waldmann Laboratory is the definition of the molecular abnormalities that drive HTLV-1 associated adult T-cell leukemia (ATL) with special emphasis on disorders of the common gamma cytokines and the JAK1/3/STAT5 pathway with implications for therapy. To perform the molecular analysis we screened the PBMCs of 36 ATL patients and 24 ethnically matched controls and identified 4 patients with activation mutations of JAK3. These somatic mutations occurred in the N-terminal (FERM) founding members (Band-4.1, Ezrin, Radixin and Moesin) domain and induced gain-of-function in JAK3. The proliferation of cells transfected with these mutant JAK3s was inhibited by the addition of tofacitinib, a JAK 2 and 3 inhibitor. A new aspect of the project involves genome-wide RNA Seq analysis of DNA from purified leukemic cells from 27 ex vivo ATL samples and 11 ATL cell lines. There were a number of interesting genes and their mutations identified including 5 Notch1 mutations supporting our previous observation of Notch mutations in ATL. There were six CARD11 missense mutations. One mutation was identical to mutations in ABC and one in GCB-DLBCL cases that is at the beginning of the coil-coil domain that has a strong gain of function. There were 4 nonsense mutations of CCR4 observed in 8 ATL samples. CCR4 is a chemokine receptor highly expressed in ATL cells and in normal Th17 and T regs. CCR4 is trans-activated by Foxp3. Nine mutations of CCR4 were all nonsense mutations that involved amino acids C329X, Q330X, and Y331X. These mutations are on the cytoplasmic tail of the 7-transmembrane chemokine receptor CCR4 and thereby would act to delete this putative activation/inhibitor region. We view this mutation as eliminating the interaction between CCR4 and beta-arrestin that acts as a normal brake on CCR4 signaling and thus the mutation represents a gain of function CCR4 in ATL. To translate these studies we have joined in a collaboration to define the effectiveness of an anti-CCR4 monoclonal antibody in the treatment of patients with severe, chronic, acute, and lymphomatous forms of ATL. To further examine molecular abnormalities including those of the JAK1/JAK3/STAT5 signaling pathway in ATL, a molecular interference screening analysis of 14 target tumor cell lines using a library of retroviral vectors for inducible expression of small hairpin shRNAs was performed in collaboration with Dr. Louis Staudt. Using this inducible shRNA loss-of-function genetic screen applied to 7 distinct cytokine dependent ATL cells lines, we demonstrated that JAK1 and JAK3 are critical for the proliferation and survival of 6 of the 7 ATL cell lines studied thereby confirming these signaling elements as targets for therapy of ATL. To further analyze the JAK1/JAK3/STAT5 pathway in ATL we have examined the ex vivo proliferation of ATL cells with special reference to disorders of the IL-2, IL-15 autocrine and the one IL-9 paracrine loop we defined previously. Initially we demonstrated that the ex vivo proliferation characteristic of ATL PBMCs could be inhibited by the simultaneous additions of antibodies to IL-2R alpha, IL-15 and IL-9. To extend our examination of the potential of JAK inhibition in the treatment of ATL, we performed experiments to evaluate the activity of the JAK2/JAK3 inhibitor, tofacitinib and the JAK1/JAK2 inhibitor, ruxolitinib on the gamma cytokine dependent ex vivo proliferation of PBMCs from patients with the smoldering and chronic type ATL. These JAK inhibitors reduced the proliferation of the ex vivo PBMCs by over 60% when given at doses achievable in vivo. To translate these preclinical demonstrations of the importance of the JAK1/JAK3/STAT5 signaling pathway in the survival of ATL malignant lymphocytes we initiated a phase II clinical trial evaluating the safety and efficacy of the JAK 1 and 2 inhibitor, ruxolitinib in patients with smoldering and chronic adult T-cell leukemia. In search for novel multicomponent combination drug therapies for ATL, in collaboration with Dr. Craig J. Thomas of the Chemical Genomics Center, NCATS/DPCI, NIH we used high-throughput robotic matrix screening of small molecules in cellular signaling analysis directed at molecular targets and pathways to identify combination therapies with active agents that demonstrate synergy. In search of synergy with the cytokine-dependent ED40515 (+) ATL cell line, the JAK1/2 inhibitor, ruxolitinib was analyzed at a range of concentrations in conjunction with a library of 466 potential therapeutic agents. Additivity/synergy was demonstrated when ruxolitinib was added in association with the Bcl-xL inhibitor (navitoclax), the HDAC inhibitor (romidepsin) and the mTOR inhibitor 1 and 2 (AZD-8055). We performed studies to define the mechanisms involved in the synergy between ruxolitinib and navitoclax. In analyzing ruxolitinib plus navitoclax we demonstrated that the combination but not the individual elements was associated with Caspase 3/7 activation and the cleavage of the anti-apoptotic molecule Mcl1 from its 40 kilodalton anti-apoptotic form to a pro-apoptotic 24 kilodalton form. We plan subsequent clinical trials involving the combination of ruxolitinib and Bcl-xL in patients with smoldering and chronic ATL.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIASC004002-44
Application #
8763672
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
44
Fiscal Year
2013
Total Cost
$1,939,404
Indirect Cost
Name
National Cancer Institute Division of Clinical Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Yeh, Chien-Hung; Bai, Xue Tao; Moles, Ramona et al. (2016) Mutation of epigenetic regulators TET2 and MLL3 in patients with HTLV-I-induced acute adult T-cell leukemia. Mol Cancer 15:15
Yeh, Chien-Hung; Bai, Xue Tao; Moles, Ramona et al. (2016) Erratum to: 'Mutation of epigenetic regulators TET2 and MLL3 in patients with HTLV-I-induced acute adult T-cell leukemia'. Mol Cancer 15:20
Sharma, Kamal; Janik, John; O'Mahony, Deidre et al. (2016) Phase II study of alemtuzumab (CAMPATH-1®) in patients with HTLV-1-associated adult T-cell leukemia/lymphoma. Clin Cancer Res :
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Yu, P; Petrus, M N; Ju, W et al. (2015) Augmented efficacy with the combination of blockade of the Notch-1 pathway, bortezomib and romidepsin in a murine MT-1 adult T-cell leukemia model. Leukemia 29:556-66
Yu, Ping; Bamford, Richard N; Waldmann, Thomas A (2014) IL-15-dependent CD8+ CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL-17 production by CD4+ T cells. Eur J Immunol 44:3330-41

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