pyrimidinones and Precursor-T-Cell-Lymphoblastic-Leukemia-Lymphoma

Topics: Amino Acid Substitution; Histiocytic Sarcoma; Humans; Imidazoles; Infant; Male; Mutation, Missense; Neoplasms, Second Primary; Oximes; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones

Inhibition of WEE1 is emerging as a promising chemosensitization strategy in many cancers including acute leukemia. Our lab and others have demonstrated that a small-molecule inhibitor of WEE1, AZD1775, sensitizes acute leukemia cells to cytarabine; however, a mechanism of combinatorial activity has remained elusive. Thus, we sought to determine the relative contribution of WEE1 targets CDK1 and CDK2 to the combinatorial activity of AZD1775 and cytarabine. To accomplish this, we expressed "WEE1 resistant" CDK1 (CDK1-AF) and CDK2 (CDK2-AF) constructs in a T-ALL cell line. Expression of CDK1/2-AF together, but neither alone, enhanced the anti-proliferative effects, DNA damage and apoptosis induced by cytarabine. Furthermore, pharmacologic inhibition of CDK1 alone or CDK1 and CDK2 together reduced the combinatorial activity of AZD1775 and cytarabine. Thus, increased activity of both CDK1 and CDK2 in response to WEE1 inhibition is necessary for the combinatorial activity of AZD1775 and cytarabine. This suggests the role of WEE1 in cells with accumulated DNA damage extends beyond regulation of CDK1 and the G2/M checkpoint and highlights the importance of WEE1 in mediating progression through the cell cycle.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Cytarabine; DNA Damage; Drug Resistance, Neoplasm; Humans; Nuclear Proteins; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones

Although in recent years, the introduction of novel chemotherapy protocols has improved the outcome of T cell acute lymphoblastic leukemia (T-ALL) patients, refractory and/or relapsing disease remains a foremost concern. In this context, a major contribution was provided by the introduction of the nucleoside analog nelarabine, approved for salvage treatment of T-ALL patients with refractory/relapsed disease. However, nelarabine could induce a life-threatening, dose-dependent neurotoxicity. To improve nelarabine efficacy, we have analyzed its molecular targets, testing selective inhibitors of such targets in combination with nelarabine.. The effectiveness of nelarabine as single agent or in combination with PI3K, Bcl2, and MEK inhibitors was evaluated on human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts. The efficacy of signal modulators in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed by flow cytometry, western blotting, and quantitative real-time PCR in T-ALL settings.. Treatment with nelarabine as a single agent identified two groups of T-ALL cell lines, one sensitive and one resistant to the drug. Whereas sensitive T-ALL cells showed a significant increase of apoptosis and a strong down-modulation of PI3K signaling, resistant T-ALL cells showed a hyperactivation of AKT and MEK/ERK1/2 signaling pathways, not caused by differences in the expression of nelarabine transporters or metabolic activators. We then studied the combination of nelarabine with the PI3K inhibitors (both pan and dual γ/δ inhibitors), with the Bcl2 specific inhibitor ABT199, and with the MEK inhibitor trametinib on both T-ALL cell lines and patient samples at relapse, which displayed constitutive activation of PI3K signaling and resistance to nelarabine alone. The combination with the pan PI3K inhibitor ZSTK-474 was the most effective in inhibiting the growth of T-ALL cells and was synergistic in decreasing cell survival and inducing apoptosis in nelarabine-resistant T-ALL cells. The drug combination caused AKT dephosphorylation and a downregulation of Bcl2, while nelarabine alone induced an increase in p-AKT and Bcl2 signaling in the resistant T-ALL cells and relapsed patient samples.. These findings indicate that nelarabine in combination with PI3K inhibitors may be a promising therapeutic strategy for the treatment of T-ALL relapsed patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arabinonucleosides; Bridged Bicyclo Compounds, Heterocyclic; Cell Survival; Drug Synergism; Humans; Phosphoinositide-3 Kinase Inhibitors; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Signal Transduction; Sulfonamides; TOR Serine-Threonine Kinases; Triazines; Tumor Cells, Cultured

While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine and that mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cytarabine; DNA Repair; Drug Synergism; Female; Flow Cytometry; Humans; Mice; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Pyrazoles; Pyrimidines; Pyrimidinones; Xenograft Model Antitumor Assays

Forodesine inhibits purine nucleoside phosphorylase, resulting in an accumulation of intracellular dGTP and consequently cell death. 9-β-D-Arabinofuranosylguanine (ara-G) is an active compound of nelarabine that is intracellularly phosphorylated to a triphosphate form, which inhibits DNA synthesis. Both agents show cytotoxicity toward T-cell malignancies. In the present study, we investigated the cytotoxicity of forodesine in vitro using ara-G-resistant leukemia cells.. T-Lymphoblastic leukemia cell line CCRF-CEM and ara-G-resistant CEM variant cell line CEM/ara-G that we had previously established were used.. A growth-inhibition assay demonstrated that CEM cells were insensitive to single-agent forodesine treatment. The cells were also insensitive to deoxyguanosine at a maximal concentration of 10 μM. CEM/ara-G cells were 80-fold more resistant to ara-G than were CEM cells, and the mode of sensitivity to forodesine and deoxyguanosine was similar to that of CEM cells. In the presence of 10 μM deoxyguanosine, forodesine effectively inhibited the growth of CEM cells but not that of CEM/ara-G cells. Flow cytometric analyses showed that combination of forodesine and deoxyguanosine induced apoptosis of CEM cells but not of CEM/ara-G cells. The addition of ara-G did not augment the cytotoxicity of the forodesine/deoxyguanosine combination towards CEM cells or CEM/ara-G cells. The combination index revealed antagonism between forodesine and ara-G. The intracellular production of ara-G triphosphate was reduced in the presence of forodesine.. Nelarabine-resistant CEM/ara-G cells are insensitive to forodesine.

Topics: Antineoplastic Agents; Arabinonucleosides; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; HL-60 Cells; Humans; Lymphoma, B-Cell; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Topics: Antineoplastic Agents; Arabinonucleosides; Cell Line, Tumor; Child; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Leukemia, Prolymphocytic, B-Cell; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Purine Nucleosides; Purines; Pyrimidinones; RNA, Messenger; RNA, Neoplasm

This article presents two case studies of patients diagnosed with T-cell acute lymphoblastic leukemia who relapsed following allogeneic hematopoietic stem cell transplantation and were subsequently enrolled in a clinical trial in which they received forodesine hydrochloride, a rationally designed, potent, transition-state inhibitor of purine nucleoside phosphorylase. Forodesine induced complete remission in both patients. Graft-versus-host disease developed subsequently but was treated successfully with conventional immunosuppressive therapy. Both patients remain in complete remission at the most recent follow-up. We hypothesize that forodesine contributed to a primary anti-leukemic cytotoxic effect as well as a secondary immunologic effect by allowing the development of an ongoing graft-versus-leukemia effect in these patients.

Topics: Adult; Antineoplastic Agents; Child, Preschool; Disease-Free Survival; Female; Graft vs Leukemia Effect; Hematopoietic Stem Cell Transplantation; Humans; Male; Neoplasm Recurrence, Local; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Pyrimidinones