Purine Analogues Research Articles

Results from the Phase 3 DUOTM Trial: A Randomized Comparison of Duvelisib Vs Ofatumumab in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

Results from the Phase 3 DUOTM Trial: A Randomized Comparison of Duvelisib Vs Ofatumumab in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma

Clofarabine-Based Reduced Intensity Conditioning Regimen with Post-Transplant Cyclophosphamide and Peripheral Stem Cell Graft in Adults with Myeloid Malignancies: Results of the Clo-Baltimore Regimen

Backgound: The Baltimore reduced-intensity conditioning (RIC) regimen using high-dose post-transplant cyclophosphamide (PTCY) is considered as a standard of care for haplo-transplant. However, it is associated with relatively low survivals (OS 46% and EFS 34% at 1 year) and high incidence of relapse (51% at 1 year), especially when considering myeloid malignancies (Luznik, 2008). Here, we have replaced fludarabine by clofarabine, a second-generation purine analogue with high anti-myeloid leukemic activity, as part of the Baltimore regimen (Clo-Baltimore regimen) and we report the results of such an approach in 36 patients with myeloid malignancies.Methods: This retrospective study included 36 adults with a median age of 60.5 (range: 31-70) years who received an allotransplant between March 2014 and March 2017. They were 18 males. Seventeen cases had acute myeloid leukemia (AML), 13 myelodysplastic syndrome (MDS), 3 myelofibrosis (MF), 1 chronic myeloid leukemia (CML), 1 mixed MDS/myeloproliferative syndrome (MPS), and 1 plasmacytoid dendritic cell neoplasm. The status at transplant was as follows: first complete remission (CR) n=16, second CR n=4, third CR n=1, active disease n=15 (MDS n=10, MF n=3; CML n=1, MDS/MPS n=1). Seven patients had already received a first allograft. Twenty-seven patients received a graft from an haploidentical donor (son n=10; daughter n=2; sister n=7; brother n=4; father n=2; nephew n=2) while 8 cases (AML n=1; MDS n=4, myelofibrosis n=2; mixed syndrome n=1) had a matched donor (sibling n=2, mud n=6) and 1 a 9/10 mis-matched unrelated donor. The Clo-Baltimore regimen consisted of Clofarabine 30 mg/m²/day days -6 to -2, cyclophosphamide 14.5 mg/kg day -6, low dose total body irradiation 2 Grays day-1, and PTCY 50 mg/kg/day days +3 and +4. All patients received PBSC as stem cell source at day 0. All patients provided informed consent for data collection before the graft.Results: Except one patient who died during aplasia of multiple organ failure (MOF), all patients engrafted. The median time of neutrophils (>1x109/L) and platelets (>50 x109/L) recoveries were 18 (range: 8-27) and 28.5 (range: 11-111) days, respectively. With a median follow-up of 12.3 months for alive patients (range: 3-39.5), 1-year and 18-month OS were both 74% (0.55-0.86), while 1-year and 18-month DFS were 74% (0.55-0.86) and 62% (0.38-0.78), respectively. At 100 days and 1 year, NRM was 5.5% and 8.3%, respectively. Seven patients have relapsed so far with a median time between graft and relapse of 90 days (range: 66-125). The 1-year relapse incidence was 19.4%. Incidences of grade 2-4 and 3-4 acute GVHD were 48% and 11.4%, respectively, while the incidence of chronic GVHD was 20% (limited 6%, extensive 14%). 1-year and 18-month GRFS were 55% (36-71) and 46% (27-64), respectively. At last follow-up, only 9 patients have died, the causes of death being relapse in 5, dermatomyositis in 1, MOF in 1, sepsis in 1 and grade 4 acute GVHD in 1.In univariate analysis, there were no differences regarding 18-month survivals between patients allografted: 1) in CR vs active disease (OS 65% vs 86%, p=0.13; DFS 56% vs 69%, p=0.18), 2)for AML vs MDS (OS 70% vs 85%, p=0.72; DFS 58% vs 85%, p=0.49), or 3) between haplo-identical vs matched+9/10 mis-matched donors (OS: 69% vs 88%, p=0.29; DFS 59% vs 61%, p=0.57).Conclusion: The Clo-Baltimore regimen has shown very encouraging results in this series for patients with myeloid malignancies, improving OS and DFS at 1 year compared to the Baltimore regimen. A low rate of relapse was observed even although using a RIC regimen, which is suitable for older patients and cases with co-morbidities. An update of the follow up will be provided at the meeting. These data have to be validated prospectively. DisclosuresTouzeau:AbbVie: Research Funding. Moreau:Onyx Pharmaceutical: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene, Janssen, Takeda, Novartis, Amgen, Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Takeda: Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria.

Negative Minimal Residual Disease Associated with Extended Response to Moxetumomab Pasudotox in Patients with Relapsed/Refractory Hairy Cell Leukemia: Long-Term Follow-up of Bone Marrow Immunohistochemistry Analyses from a Phase 1 Study

The clinical significance of minimal residual disease (MRD) in patients with relapsed/refractory hairy cell leukemia (r/r HCL) who have achieved complete response (CR) has not been demonstrated. In other hematologic malignancies, MRD eradication may be associated with prolonged clinical response (Hourigan et al. Leukemia 2017;31:1482-90; Lahuerta et al. J Clin Oncol 2017;epub ahead of print). Moxetumomab pasudotox (formerly CAT-8015), a recombinant immunotoxin fusion protein that specifically binds to B lymphocyte-specific surface antigen CD22, has been shown by flow cytometry to eradicate MRD in a significant (P= 0.04) percentage of patients with r/r HCL (Kreitman et al. ASCO 2015). However, a detailed study of MRD by bone marrow immunohistochemistry (IHC) was not previously presented. We analyzed data from a phase 1 dose escalation study of moxetumomab pasudotox in r/r HCL patients to test the hypothesis that eradication of MRD in bone marrow, assessed by IHC, may be associated with prolonged remission.CAT-8015-1001 (NCT00586924) was a phase 1 study evaluating the safety and efficacy of moxetumomab pasudotox in patients with r/r HCL who previously had received at least 2 courses of treatment, at least 1 of which included a purine analog; initial results from the dose escalation phase in 28 patients have been reported (Kreitman et al. J Clin Oncol 2012;30:1822-8). Moxetumomab pasudotox was administered on days 1, 3, and 5 of each 28-day cycle until 2 cycles past complete remission (CR), disease progression, unacceptable toxicity, or neutralizing antibodies. In the dose escalation phase, 28 patients received moxetumomab pasudotox 5, 10, 20, 30, 40, or 50 µg/kg. An additional 21 patients were enrolled in the 50-µg/kg cohort.Bone marrow samples were obtained at baseline and at multiple time points during and after treatment. A central pathology laboratory performed hematoxylin and eosin and IHC staining for HCL/B-cell antigens CD20, CD79a, Annexin A1, DBA.44, and PAX-5. A blinded independent central pathologist reviewed (BICR) and assessed stained slides as IHC MRD+ or IHC MRD− for each time point.The best BICR IHC MRD response was compared with the best investigator-reported (IR) disease response. Duration of CR, duration of response (DOR), progression-free survival (PFS), and overall survival were evaluated for patients with a best response of BICR IHC MRD+ vs MRD−.Thirty-seven patients were evaluated for BICR IHC MRD; posttreatment bone marrow biopsies were not available for 11 patients, and biopsies from one patient were non-evaluable. The BICR IHC MRD status appears to be associated with the IR response; 17 of 18 BICR IHC MRD− patients had a best response of CR vs 9 of 19 MRD+ patients. Among patients found to be BICR IHC MRD− at 1 or more posttreatment evaluations, median duration of CR and median PFS were not reached after 84 months of follow-up vs 13.08 and 82.07 months, respectively, for patients who were consistently MRD+ at all time points. DOR was 82.7 vs 54.7 months for the BICR IHC MRD− and MRD+ groups, respectively.Cladribine monotherapy and cladribine followed by rituximab eliminated IHC MRD in some newly diagnosed HCL patients (Sigal et. al, Blood 2010;115:1893-6; Ravandi et. al Blood 2011;118:3818-23). Although vemurafenib demonstrated 96% to 100% overall response rates and 35% to 42% CR rates in patients with r/r HCL, all CR patients were IHC MRD+, and duration of MRD+ CRs was 19 months (Tiacci et al. N Engl J Med 2015;373:1733-47). Moxetumomab pasudotox is, to our knowledge, the only non-chemotherapy regimen shown to eradicate MRD in a meaningful percentage of patients with r/r HCL. Here, BICR findings confirmed elimination of IHC MRD in 49% of evaluable patients. MRD− patients experienced a statistically significantly longer duration of CR (P=0.0002) and PFS (P=0.0031) vs MRD+ patients.Based on the results of this retrospective analysis, together with observations made in the vemurafenib study, BICR IHC MRD− vs MRD+ status is associated with extended DOR and PFS in patients with r/r HCL treated with moxetumomab pasudotox. If this finding can be replicated in additional clinical studies, including the phase 3 pivotal study of moxetumomab pasudotox, IHC MRD determinations may be useful in evaluating the efficacy of therapies in the r/r HCL population. [Display omitted] DisclosuresKreitman:NIH: Patents & Royalties: Co-inventor on pending Moxetumomab Pasudotox patent owned by NIH. Robak:Akari Therapeutics Plc: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding. Coutre:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy; Novartis: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Marshall:MedImmune: Employment. Yang:MedImmune: Employment, Equity Ownership, Other: Travel expenses. Pastan:NIH: Patents & Royalties: Pastan is an inventor on several patents on immunotoxins that have all been assigned to the NIH. Yao:MedImmune: Employment, Equity Ownership.

CDK9 Inhibition with LDC526 Is an Effective Treatment for T-Cell Prolymphocytic Leukemia (T-PLL) in Vitro

▪IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare leukemia with an aggressive disease course, characterized by chemotherapy resistance and a median overall survival of less than two years. Apart from defects in DNA damage response genes like ATM and genes of JAK/STAT signaling, overexpression of MYC, often caused by gain of chromosomal material, is frequently observed in T-PLL. Intravenous application of the anti-CD52 antibody Alemtuzumab has been demonstrated to improve progression-free survival and is currently considered the standard of care. In younger patients remissions can be consolidated by allogeneic stem cell transplantation, which results in long-term remission in a small subgroup of patients. The prognosis in the majority of patients relapsing from their disease is dismal and thus novel treatment options are urgently needed. Cyclin-dependent kinase 9 (CDK9), a serine/ threonine kinase which is ubiquitously expressed, is the catalytic unit of the positive transcription elongation factor (P-TEFb). In the presence of cyclin T, p-TEFb stimulates transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA Pol II). CDK9 inhibition by LDC526, a specific CDK9 inhibitor, leads to reduced phosphorylation of CTD-Ser2, Ser5 and Ser7 of RNA Pol II and subsequently to the downregulation of MYC and MCL-1.Methods</spanCD3+CD4+ tumor cells from cryopreserved peripheral blood samples of 11 T-PLL patients were enriched to a purity of >95% by fluorescence-activated cell sorting (FACS) or magnetic activated cell separation (MACS). RNA and DNA were isolated using the RNeasy Mini Kit® or the QIAamp DNA blood midi kit® (Qiagen, Hilden, Germany), respectively. To determine IC50 values, we performed cell viability assays for T-PLL cells of 11 patients treated with LDC526 and a panel of reference compounds compared to DMSO controls (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Fitchburg, WI, USA). Gene expression profiling was performed in primary T-PLL cells treated with the CDK9 inhibitor LDC526 in a concentration of 10µM compared to DMSO (n=3) on Clariom S Pico Assays (Affymetrix, Santa Clara, CA, USA) and analyzed by gene set enrichment analysis (GSEA) and GeneTrail analysis. Gene expression measurement was performed with quantitative real-time PCR (qPCR) for MYC (Taqman, Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was analyzed for pCTD Ser2, Ser5 and Ser7, MYC, p-STAT3 and MCL-1 by Western Blot (n=4) after LDC526-treatment in three different concentrations (10, 30 and 100 µM).ResultsLDC526 inhibited T-PLL cell survival in vitro in a dose-dependent manner. The median IC50 was 1.7 µM (range: 0.9 -3.5) which compared favourably to a panel of reference compounds including fludarabine, clofarabine, methotrexate, cytarabine and ruxolitinib (median IC50 values 6.4, 6.2, 29.9, 8.3, and 29.2, respectively). Similar experiments with BAY1143572, a structurally closely related CDK9 inhibitor, which is currently investigated in phase I studies of acute myeloid leukemia, revealed an even lower median IC50 of 0.9 µM (range: 0.5 - 1.7). Of note, the purine analogues fludarabine and clofarabine were only active in some of the patients tested, whereas both CDK9 inhibitors showed activity in all samples analyzed.Gene expression profiles of LDC526 treated T-PLL cells compared to DMSO controls revealed an enrichment of genes in gene sets associated with interferon and TNFA signatures, MYC targets as well as JAK/STAT genes, which were subsequently confirmed by qPCR experiments. At the protein level LDC526 dose-dependently reduced phosphorylation of CTD-Ser2, Ser7 and to a lesser extent of Ser5 compared to DMSO controls resulting in efficient down-regulation of MYC, MCL-1 and p-STAT3.ConclusionsOur experiments identify CDK9 as a novel therapeutic target in T-PLL which can be efficiently inhibited by LDC526 and BAY1143572 at clinically meaningful concentrations. CDK9 inhibition resulted in both reduced T-PLL cell survival and down-regulation of MYC and JAK/STAT signaling, which have a key role in the molecular pathogenesis of T-PLL. DisclosuresDürig:Lead Discovery Center: Research Funding.

Clofarabine/Busulfan-Based Reduced Intensity Conditioning Regimens Provides Very Good Survivals in Patients Allografted in Remission for Acute Myeloid Leukemia: A Retrospective Study on Behalf of the SFGM-TC

Introduction: Clofarabine/busulfan based reduced conditioning regimens (RIC) have been developed recently with the hope to improve outcomes in older patients allografted for myeloid malignancies. Indeed, clofarabine, a second generation purine analogue, has been proved to have higher anti-leukemic myeloid activity compared to fludarabine, a drug extensively used as part of RIC for allogeneic stem cell transplantation (allo-SCT). However, larger studies are needed to confirm these results.Method: This was a retrospective study including all patients who received a clofarabine/busulfan based RIC allo-SCT for myeloid malignancies and reported within the SFGM-TC registry. Data were obtained through PROMISE, an internet-based system shared by all European transplantation centers. All patients gave informed consent allowing to collect their personal data from this data base.RIC regimen consisted of clofarabine 30 mg/m²/day 4 to 5 days (Clo), busulfan 3.2 mg/kg/day 2 days (B2) and one or two days of rabbit antithymocyte globulin (A1 or A2). CloB2A2 have been used first as a conditioning platform while some centers have used CloB2A1 since 2014 in order to try to decrease relapse and improve the results of the CloB2A2 patients.The primary objective of the study was to report the main outcomes of the whole cohort at 2 years: overall survival (OS), disease free survival (DFS), relapse incidence (RI), non-relapse mortality (NRM) and GVHD and Relapse-free survival (GRFS). Secondary objectives were to identify prognostic factors and to compare outcomes between CloB2A2 (n=43)/CloB2A1 (n=41) cases.Results: Between January 2008 and December 2016, 84 patients (males n=47, median age 61.6 years) met the inclusion criteria. The majority of patients had acute myeloid leukemia (AML, n=63) while 21 cases had myelodysplastic or myeloproliferative syndrome. Sixty-one patients were in complete remission (CR) at time of transplant (CR1 n=49. CR2, n=12), including 55 AML cases (CR1 n=43; CR2 n=12). All patients but one (bone marrow graft) received peripheral blood stem cell as graft source. All patients received matched donors (sibling n=34; unrelated n=50). There were no significant differences in term of characteristics between CloB2A2 (n=43, median follow-up 39 months) and CloB2A1 (n=41, median follow-up 21 months) patients, except the median age of the cohort which was higher in the latter group (65 vs 61 years, p=0.02) and, as expected, the median year of transplant (2013 vs 2015, p<0.001). All patients engrafted except one CLOB2A1 case.With a median follow up of 31 months (range: 5.7-74.1) for patients alive, 2-year OS, DFS, RI, NRM and GRFS were 64.5% (53.8-75.2); 57.2% (46.2-68.2); 27.7% (18.2-37.9); 15.1% (8.2-23.9) and 43.6% (32.5-54.7), respectively, for the whole cohort. Considering AML in remission, 2-year OS, DFS, RI, NRM and GRFS were 74.2% (62-86.5); 66.8% (53.6-79.9); 23.4% (12.7-36); 9.8% (3.5-19.9) and 50.9% (36.9-64.9), respectively.2-year outcomes were similar between CloB2A1 and CloB2A2 sub-groups: OS 61.2% (43.8-78.6) vs 65.1% (50.9-79.4), p=0.93; DFS 60.8% (44.9-76.6) vs 54.7% (39.6-69.7), p=0.93; RI 28.8% (15.1-44) vs 26.3% (14-40.3), p=0.51; NRM 10.5% (3.2-22.7) vs 19% (8.8-32.2), p=0.55; GRFS 45% (27.9-62) vs 41.7% (27-56.5), p=0.96. Comparable outcomes were also observed when considering only AML patients in CR. However, a trend for lower NRM within the CloB2A1 subgroup was observed (4.2% (0.3-18.1) vs 16% (4.8-33), p= 0.14).In multivariate analysis, active disease at transplant was the only factor adversely impacting 2 years outcomes.Conclusion: CloB2A2/A1 RIC regimens provides very good results for AML patients allografted in CR. CloB2A1 should be retained as a RIC platform for these patients. Our results have to be confirmed prospectively. DisclosuresPeffault De Latour:Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Mohty:Sanofi: Honoraria, Speakers Bureau.

Enhanced Safety and Tolerability of Dose Intensive Pentostatin and Rituximab in Patients with CLL or Low-Grade B-Cell NHL.

BackgroundRecent studies have suggested that combination chemotherapy with a purine analog and a monoclonal antibody may have additive as well as synergistic activity in the treatment of NHL and CLL. The first immunochemotherapeutic regimen combined fludarabine (F) and rituximab (R). Although it was efficacious, it was also myelotoxic and immunosuppressive. This multicenter Phase II trial investigated the efficacy and safety of another purine analog, pentostatin (P), in combination with R in the treatment of NHL and CLL patients. (Some of this data has been presented at the 2005 Pan-Pacific Lymphoma Conference).MethodsOne hundred forty-seven patients were enrolled in this study. Eligibility criteria allowed previously treated (PT) and treatment naïve (UT) patients diagnosed with low-grade NHL (REAL classification) or stage II, III, or IV CLL (mod. Rai staging). Treatment consisted of intravenous infusions of R (375 mg/m2) on day 1 and R (375 mg/m2) plus dose intensive P (4 mg/m2) on days 8, 15, 22, 36, 43, and 50, completing 1 cycle. Clinical evaluation was performed after 60 days. If complete response or progressive disease was documented, the treatment was stopped. All other patients were eligible to then receive cycle 2. Patients were stratified by disease and by prior treatment status.ResultsOne hundred thirty three patients were eligible for evaluation. One hundred sixty six cycles were administered. Response rate (RR) for NHL-PT was 51% and for NHL-UT was 78%. RR for CLL-PT was 34% and for CLL-UT 42%. There were 30 NHL patients eligible for cycle 2 and 22 went on to receive the additional cycle. Ten patients (45%) achieved an improved objective response. Seventeen CLL patients were eligible for cycle 2 and 8 completed cycle 2. There were 2 (25%) patients that achieved an improved objective response. Median duration of response (DoR) was 11.7, 21.5, 11.9, and 23.7 months, respectively. Median progression-free survival (PFS) was 14.8, 23.5, 13.9, and 25.6 months, respectively. There was a single case of grade 4 neutropenia in each of the PT and UT groups. Overall, neutropenia was the most common adverse event (AE) (6%) in the PT group and was the most common hematological AE (7%) in the UT group. Four patients died within 30 days of receiving chemotherapy (age range 64–88). Causes of death were respiratory insufficiency (2), MI/CAD, and sepsis/CLL.ConclusionsThis immunochemotherapeutic regimen of P+R is active in previously untreated and treated patients with indolent NHL or stage II–IV CLL. The objective response can be improved with prolonged therapy. With only 2/133 evaluable patients experiencing Gr 4 neutropenia, we have demonstrated that this dose intensive regimen was very safe and well tolerated. Future trials comparing the tolerability and efficacy of P+R in combination with cyclophosphamide and/or mitoxantrone to other standard regimens are warranted. The use of combination therapy in elderly patients should be used with caution due to associated co-morbidities.

A Multicenter Experience with Single Agent Bortezomib in Non-Hodgkin's Lymphoma Reveals Marked Differences in Sub-Type Sensitivity to Proteasome Inhibition.

To date, we have treated 51 patients (pts.) on an ongoing multicenter phase II study of botezomib in pts. with indolent NHL and mantle cell lymphoma (MCL). The average number of cycles received per patient was four. This includes 19 patients with follicular lymphoma (FL), 23 with MCL, 5 with small lymphocytic lymphoma-CLL type; and 4 with marginal zone lymphoma (MZL). All patients were required to sign an informed consent and have adequate hematologic counts (i.e. an ANC of > 1500 cells/ml and a platelet count > 50,000/ml). All but one patient had received some form of treatment prior to receiving bortezomib, including: CHOP +/− R (60%); CVP +/− R (20%); or some other purine analog based treatment program (15%). Patients were treated at a dose of 1.5 mg/m2 on Days 1, 4, 8 and 11 every 21 days. No Grade IV toxicities were noted, and the most common Grade 3 toxicity was lymphopenia. Three pts. experienced grade 3 sensory neuropathy. Re-staging studies were routinely performed every two cycles. The overall response rate was 55%, though there is considerable variability among the subtypes both in the response rate, and time to response. One of 5 pts. with SLL experienced a partial remission, in contrast to all 4 patients with marginal zone lymphoma who experienced a partial remission. In most of these latter cases, patients experienced tumor shrinkage after 2 to 4 doses. While all pts. are in active follow-up, one has sustained the response in excess of 10+ months, the other in excess of 18+ months. Of 23 pts. with MCL, the response rate among the evaluable pts. is 56%. While most of these pts. are in active follow-up, the shortest duration of remission was 6 months, the longest 19 months. Patients typically responded by the second cycle, or didn't respond. Two patients have received 3 additional courses of therapy, achieving a PR on each re-treatment. In FL the response rate is 60%, including one CR and one Cru. These pts. responded later in treatment, typically after the third cycle and later. To date, the median duration of response has not been reached. These data continue to support the activity of bortezomib in pts. with select sub-types of NHL, and raise the merits of potential re-treatment in responding pts.

Clofarabine induces ERK/MSK/CREB activation through inhibiting CD99 on Ewing sarcoma cells.

Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine’s conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation.

Synthesis and screening of 6-alkoxy purine analogs as cell type-selective apoptotic inducers in Jurkat cells.

Purines are ubiquitous structures in cell biology involved in a multitude of cellular processes, because of which substituted purines and analogs are considered excellent scaffolds in drug design. In this study, we explored the key structural features of a purine-based proapoptotic hit, 8-tert-butyl-9-phenyl-6-benzyloxy-9H-purine (1), by setting up a library of 6-alkoxy purines with the aim of elucidating the structural requirements that govern its biological activity and to study the cell selectivity of this chemotype. This was done by a phenotypic screening approach based on cell cycle analysis of a panel of six human cancer cell lines, including T cell leukemia Jurkat cells. From this study, two derivatives (12 and 13) were identified as Jurkat-selective proapoptotic compounds, displaying superior potency and cell selectivity than hit 1.

Targeting glioma cells by antineoplastic activity of reversine.

Gliomas are the most common type of primary central nervous system tumors and despite great advances in understanding the molecular basis of the disease very few new therapies have been developed. Reversine, a synthetic purine analog, is a multikinase inhibitor that targets aurora kinase A (AURKA) and aurora kinase B (AURKB). In gliomas, a high expression of AURKA or AURKB is associated with a malignant phenotype and a poor prognosis. The present study investigated reversine-related cellular and molecular antiglioma effects in HOG, T98G and U251MG cell lines. Gene and protein expression were assessed by reverse transcription-quantitative PCR and western blotting, respectively. For functional assays, human glioma cell lines (HOG, T98G and U251MG) were exposed to increasing concentrations of reversine (0.4–50 µM) and subjected to various cellular and molecular assays. Reversine reduced the viability and clonogenicity in a dose- and/or time-dependent manner in all glioma cells, with HOG (high AURKB-expression) and T98G (high AURKA-expression) cells being more sensitive compared with U251MG cells (low AURKA- and AURKB-expression). Notably, HOG cells presented higher levels of polyploidy, while T98G presented multiple mitotic spindles, which is consistent with the main regulatory functions of AURKB and AURKA, respectively. In molecular assays, reversine reduced AURKA and/or AURKB expression/activity and increased DNA damage and apoptosis markers, but autophagy-related proteins were not modulated. In conclusion, reversine potently induced mitotic catastrophe and apoptosis in glioma cells and higher basal levels of aurora kinases and genes responsive to DNA damage and may predict improved antiglioma responses to the drug. Reversine may be a potential novel drug in the antineoplastic arsenal against gliomas.

Synthesis of Purine Analogues: Photocatalyst-Free Visible-Light-Enhanced Annulation Approach to Pyrazolo[1,5-a][1,3,5]triazine-2,4-diamines

A new photocatalyst-free visible-light-enhanced strategy for the synthesis of pyrazolo[1,5-a][1,3,5]triazine-2,4-diamines via the formation of electron donor-acceptor (EDA) complexes is reported. The in situ generated pyrazolthiourea intermediates from 1H-pyrazol-3-amines and isothiocyanates undergo formal [4 + 2] annulation with 1,1,3,3-tetramethylguanidines (TMG) to deliver the corresponding products involved in three C-N bond formations in a one-pot protocol. The formation of EDA complex from pyrazolthiourea and TMG is confirmed by UV-vis spectroscopy and 1H NMR experiments. Moreover, this mild reaction proceeds in the absence of any external transition metals, oxidants, bases, and ligands. This efficient methodology for the synthesis of purine analogues pyrazolo[1,5-a][1,3,5]triazine-2,4-diamines provides potential synthetic applications in the field of drug research and development.

A Drug Repositioning Approach Identifies a Combination of Compounds as a Potential Regimen for Chronic Lymphocytic Leukemia Treatment.

Drug repositioning is a promising and powerful innovative strategy in the field of drug discovery. In this study, we screened a compound-library containing 800 Food and Drug Administration approved drugs for their anti-leukemic effect. All screening activities made use of human peripheral blood mononuclear cells (PBMCs), isolated from healthy or leukemic donors. Compounds with confirmed cytotoxicity were selected and classified in three groups: i) anti-neoplastic compounds which are drugs used in leukemia treatment, ii) compounds known to have an anti-cancer effect and iii) compounds demonstrating an anti-leukemic potential for the first time. The latter group was the most interesting from a drug repositioning perspective and yielded a single compound, namely Isoprenaline which is a non-selective β-adrenergic agonist. Analysis of the cytotoxic effect of this drug indicated that it induces sustainable intracellular ATP depletion leading, over time, to necrotic cell death. We exploited the Isoprenaline-induced intracellular ATP depletion to sensitize primary leukemic cells to fludarabine (purine analogue) and Ibrutinib (Bruton’s tyrosine kinase inhibitor) treatment. In-vitro treatment of primary leukemic cells with a combination of Isoprenaline/fludarabine or Isoprenaline/Ibrutinib showed a very high synergistic effect. These combinations could constitute a new efficient regimen for CLL treatment following successful evaluation in animal models and clinical trials.

Leucémie à tricholeucocytes : quelles sont les meilleures options thérapeutiques pour les patients en rechute ou réfractaires ?

Hairy cell leukemia is a rare form of leukemia: three hundred new cases are diagnosed each year in France. The diagnosis is based on: (1) morphological examination of the blood and bone marrow smear, (2) analysis by flow cytometry of hairy cells, which express three or the four following markers: CD11c, CD25, CD103 and CD123, (3) identification of the BRAFV600E mutation, a true molecular marker of the disease. The management of treatment has evolved considerably in recent years. As of today, the purine analogues remain the standard treatment in the first line. Relapses are however observed in about 40% of cases. In the event of a first relapse, the preferred option is treatment with immunochemotherapy i.e. a combination of cladribine plus rituximab. Subsequent relapses are treated with moxetumomab pasudotox or BRAF inhibitors which provide indisputable benefits if third-line treatment is required. We will discuss in patients with relapsed/refractory hairy cell leukemia the needs for personalized medicine and the advantages and disadvantages of each treatment modality. The good prognosis for LT requires treatments that are not immunosuppressive, non-myelotoxic, and do not increase the risk of secondary cancers.

Photophysical properties of fluorescent nucleobase P-analogues expected to monitor DNA replication

In this work, we computationally design a series of fluorescent purine analogues based on the 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (P) to monitor the DNA replication process with merely a minimal perturbation to the natural structure of nucleic acid. The P-modified fluorescent probes present red-shifted absorption spectra and enhanced photoluminescence due to the additional π-conjugation resulting from the fluorophore modification and the ring-expansion. Efficient fluorescence quenching of P-analogues occurs upon pairing with the complementary 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (Z) due to the nonradiative relaxation from the low-lying dark excited state to the ground state of Z moiety. Especially, the P3 and the P7, which have high fluorescence intensity in both gas and liquid phases, are proposed as the sensors for studying conformational switching in the presence and absence of a complementary sequence. Also examined are the influences of hydration and the linking to deoxyribose on absorption and emission processes. Besides, the potential phosphorescence emission of these modified base pairs is taken into account by constructing the relaxed potential energy curves of S0, T1 and S1 states.

Leucémie à tricholeucocytes

AbstractHairy cell leukemia is a rare form of leukemia: three hundred new cases are diagnosed each year in France. The diagnosis is based on: 1) morphological examination of the blood and bone marrow smear, 2) analysis by flow cytometry of hairy cells, which express three or the four following markers: CD11c, CD25, CD103 and CD123, 3) identification of the BRAFV600E mutation, a true molecular marker of the disease.The management of treatment has evolved considerably in recent years. As of today, the purine analogues remain the standard treatment in the first line. Relapses are however observed in about 40% of cases. In the event of a first relapse, the preferred option is treatment with immunochemotherapy i.e. a combination of cladribine plus rituximab. Subsequent relapses are treated with moxetumomab pasudotox or BRAF inhibitors which provide indisputable benefits if third-line treatment is required. We will discuss in patients with relapsed/refractory hairy cell leukemia the needs for personalized medicine and the advantages and disadvantages of each treatment modality.The good prognosis of hairy cell leukemia requires treatments that are not immunosuppressive, non-myelotoxic, and do not increase the risk of secondary cancers.

Fludarabine Inhibits Infection of Zika Virus, SFTS Phlebovirus, and Enterovirus A71.

Viral infections are one of the leading causes in human mortality and disease. Broad-spectrum antiviral drugs are a powerful weapon against new and re-emerging viruses. However, viral resistance to existing broad-spectrum antivirals remains a challenge, which demands development of new broad-spectrum therapeutics. In this report, we showed that fludarabine, a fluorinated purine analogue, effectively inhibited infection of RNA viruses, including Zika virus, Severe fever with thrombocytopenia syndrome virus, and Enterovirus A71, with all IC50 values below 1 μM in Vero, BHK21, U251 MG, and HMC3 cells. We observed that fludarabine has shown cytotoxicity to these cells only at high doses indicating it could be safe for future clinical use if approved. In conclusion, this study suggests that fludarabine could be developed as a potential broad-spectrum anti-RNA virus therapeutic agent.

Recent Understanding and Future Directions of Recurrent Corticotroph Tumors

Corticotroph tumors (CTs) are pituitary neoplasms arising from the Tpit lineage, which may or not express adrenocorticotrophic hormone (ACTH). Functioning CTs cause Cushing’s disease (CD), which has high morbidity and mortality due to hypercortisolemia. “Non-functioning” or silent CTs (SCT) and the Crooke’s cell subtypes do not cause CD and may be asymptomatic until manifested by compressive symptoms and are more frequently found as macroadenoma. Both tend toward more aggressive behavior, recurrence, and a higher rate of malignant transformation to pituitary carcinoma. Tumorigenesis involves genetic, epigenetic, and post-transcriptional disruption of cell-cycle regulators, which increase cell proliferation, POMC overexpression, ACTH transcription, and/or hypersecretion. Furthermore, functioning CTs develop resistance to glucocorticoid-mediated negative feedback on ACTH secretion, through increased expression of testicular orphan nuclear receptor 4 (TR4), heat-shock protein 90 (HSP90), and loss-of-function mutation of CDK5 and ABL enzyme substrate 1 (CABLES1) gene. Overt autonomous hypercortisolemia is difficult to control, and multiple diagnostic studies and therapeutic modalities are commonly required. Cell-cycle regulation depends mainly on p27, cyclin E, cyclin-dependent kinases (CDKs), and the retinoblastoma protein (Rb)/E2F1 transcription factor complex. Gain-of-function mutations of ubiquitin-specific protease (USP) 8, USP48, and BRAF genes may subsequently cause overexpression of epithelial growth factor receptor (EGFR), and enhance POMC transcription, cell proliferation, and tumor growth. Epigenetic changes through micro RNAs and decreased DNA deacetylation by histone deacetylase type 2 (HDAC2), may also affect tumor growth. All the former mechanisms may become interesting therapeutic targets for CTs, aside from temozolomide, currently used for aggressive tumors. Potential therapeutic agents are EGFR inhibitors such as gefitinib and lapatinib, the purine analog R-roscovitine by dissociation of CDK2/Cyclin E complex, the HSP90 inhibitor silibinin (novobiocin), to reduce resistance to glucocorticoid-mediated negative feedback, and BRAF inhibitors vemurafenib and dabrafenib in BRAF V600E positive tumors. This review summarizes the molecular mechanisms related to CTs tumorigenesis, their diagnostic approach, and provides an update of the potential novel therapies, from the lab bench to the clinical translation.

Multicenter comparison of high-dose cytarabine-based regimens versus liposomal daunorubicin and cytarabine (CPX-351) in patients with secondary acute myeloid leukemia

Liposomal daunorubicin/cytarabine (CPX-351) gained FDA approval for secondary AML after demonstrating improved outcomes over daunorubicin and cytarabine (7 + 3). A number of study limitations prompted a comparison of safety/efficacy of CPX-351 against regimens containing a purine analogue and high-dose cytarabine (HIDAC). This retrospective study compared complete response rates with/without count recovery (CR/CRi) between HIDAC-based regimens and CPX-351 in 169 patients with newly diagnosed sAML. The CR/CRi rate was 62.7% in the HIDAC-based therapy arm vs. 47.9% in the CPX-351 arm (p = 0.002 [one-sided for non-inferiority]). Median time to absolute neutrophil and platelet count recovery was shorter after HIDAC-based therapy (18 and 23 days, respectively) compared to CPX-351 (36 and 38 days; p < 0.001). Median overall survival was 9.8 months in the HIDAC-based group and 9.14 months in the CPX-351 group. 30-day mortality was greater with CPX-351 (8.5%) compared to HIDAC-based (1.3%; p = 0.039). These results reveal comparable efficacy and favorable safety with HIDAC-based regimens.

Coagulase-negative staphylococci release a purine analog that inhibits Staphylococcus aureus virulence

Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.

Phase 2 study of ibrutinib in classic and variant hairy cell leukemia

Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.