Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells.

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

Chk1 inhibition significantly potentiates activity of nucleoside analogs in TP53-mutated B-lymphoid cells.

Treatment options for TP53-mutated lymphoid tumors are very limited. In experimental models, TP53-mutated lymphomas were sensitive to direct inhibition of checkpoint kinase 1 (Chk1), a pivotal regulator of replication. We initially tested the potential of the highly specific Chk1 inhibitor SCH900776 to synergize with nucleoside analogs (NAs) fludarabine, cytarabine and gemcitabine in cell lines derived from B-cell malignancies. In p53-proficient NALM-6 cells, SCH900776 added to NAs enhanced signaling towards Chk1 (pSer317/pSer345), effectively blocked Chk1 activation (Ser296 autophosphorylation), increased replication stress (p53 and γ-H2AX accumulation) and temporarily potentiated apoptosis. In p53-defective MEC-1 cell line representing adverse chronic lymphocytic leukemia (CLL), Chk1 inhibition together with NAs led to enhanced and sustained replication stress and significantly potentiated apoptosis. Altogether, among 17 tested cell lines SCH900776 sensitized four of them to all three NAs. Focusing further on MEC-1 and co-treatment of SCH900776 with fludarabine, we disclosed chromosome pulverization in cells undergoing aberrant mitoses. SCH900776 also increased the effect of fludarabine in a proportion of primary CLL samples treated with pro-proliferative stimuli, including those with TP53 disruption. Finally, we observed a fludarabine potentiation by SCH900776 in a T-cell leukemia 1 (TCL1)-driven mouse model of CLL. Collectively, we have substantiated the significant potential of Chk1 inhibition in B-lymphoid cells.

Ofatumumab added to dexamethasone in patients with relapsed or refractory chronic lymphocytic leukemia: Results from a phase II study.

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high-dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab-dexamethasone (O-Dex) combination in relapsed or refractory CLL. The trial was an open-label, multicenter, nonrandomized, Phase II study. The O-Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2-6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1-4 and 15-18; Cycles 1-6). The O-Dex regimen was given until best response, or a maximum of six cycles. Thirty-three patients (pts) were recruited. Twenty-four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression-free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3-5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O-Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101).

TP53 mutation analysis in chronic lymphocytic leukemia: comparison of different detection methods.

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: a prospective analysis.

Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p=0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period.

Complex karyotype and translocation t(4;14) define patients with high-risk newly diagnosed multiple myeloma: results of CMG2002 trial.

The prognostic impact of chromosomal abnormalities was evaluated by fluorescence in situ hybridization with cytoplasmic immunoglobulin light chain staining (cIg-FISH) and by classical metaphase cytogenetics in a cohort of 207 patients with newly diagnosed multiple myeloma who were treated with high-dose therapy followed by autologous stem cell transplantation in the CMG2002 clinical trial. The incidence of chromosomal abnormalities detected by FISH was as follows: 52.7% for del(13)(q14), 6.5% for del(17)(p13), 18.6% for t(11;14)(q13;q32), 22.8% for t(4;14)(p16;q32) and 45.7% for gain(1)(q21). Metaphase cytogenetic analysis revealed a complex karyotype in 19.1% and hyperdiploidy in 21.7% of patients. The overall response rate was not influenced by the presence of any studied chromosomal abnormality. Patients with a complex karyotype, those with translocation t(4;14) and those with gain of the 1q21 locus had a shorter time to progression (TTP) and overall survival (OS). Other genomic changes such as translocation t(11;14) and del(13q) had less impact on TTP and OS. In multivariate analysis, complex karyotype, translocation t(4;14) and ß(2)-microglobulin level > 2.5 mg/L were independent prognostic factors associated with poor overall survival. Their unfavorable prognostic impact was even more pronounced if they were present in combination. Patients with t(4;14) present together with a complex karyotype had the worst prognosis, with a median OS of only 13.2 months, whereas patients with a normal karyotype or karyotype with ≤ 2 chromosomal changes had the best outcome, with 3-year OS of 85.9%. In conclusion, complex karyotype, gain of 1q21 region and translocation t(4;14) are major prognostic factors associated with reduced survival of patients with newly diagnosed multiple myeloma treated with autologous stem cell transplantation.

Prognosis of children with mixed phenotype acute leukemia treated on the basis of consistent immunophenotypic criteria.

BACKGROUND: Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines. DESIGN AND METHODS: Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed. RESULTS: The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53+/-10% and 76+/-2% at 5 years, respectively, P=0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts. CONCLUSIONS: Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.

Characterization of a GM7 glioblastoma cell line showing CD133 positivity and both cytoplasmic and nuclear localization of nestin.

A newly established GM7 cell line was derived from the tumor tissue of a 65-year-old man surgically treated for a relapse of glioblastoma multiforme that occurred 10 months after first surgery following radiotherapy. GM7 cells exhibit spindle or glia-like morphology, and multinucleated giant cells are also present in the culture. The cells proliferate rapidly (PDT is about 18 h) and tend to grow in multilayer without contact inhibition. Using G-banding and SKY, the GM7 cell line was identified as near-triploid with a large number of structural and numerical abnormalities. Repeated karyotyping during long-term cultivation confirmed a chromosome number of 70+/-3 chromosomes per cell. Special attention was paid to the immunocytochemical analysis of protein markers in this cell line; GM7 cells showed strong positivity for CD133, vimentin, nestin, NF-160 and S-100 protein and weak positivity for GFAP and NSE, but were negative for synaptophysin. The most important features of the GM7 cell line are its stable phenotype CD133+/nestin+, which are accepted as stem cell markers in neural stem/progenitor cells, and especially unusual intracellular localization of the IF protein nestin, which was detected and repeatedly confirmed both in the cytoplasm and cell nucleus. For this reason, the new GM7 glioblastoma cell line represents an important model suitable not only for further studies on glioblastoma biology and cancer stem cells, but particularly for the detailed investigation of the role of nestin in transformed cells.

Treatment of chronic myeloid leukemia with autologous transplantation using peripheral blood stem cells or bone marrow cultured in IL-2 followed by IL-2, GM-CSF, and IFN-alpha administration.

Interleukin-2 (IL-2) is able to generate nonspecific cytotoxic effectors from hematopoietic precursors. We evaluated the feasibility and efficacy of chronic myeloid leukemia (CML) treatment with autologous hematopoietic stem cell transplantation (HSCT) using grafts cultured in IL-2 followed by immunotherapy with IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-alpha. Eight patients with CML were enrolled: five in an accelerated phase and three in a chronic phase. They received peripheral blood stem cells (PBSC) or bone marrow (BM) cultured in a medium containing IL-2 for 24 h. A median of 1.29 x 10(6) CD34+ cells/kg were infused after conditioning with busulfan (12 16 mg/kg) in PBSC recipients. BM was infused without prior myeloablative therapy. The engraftment occurred with a median of 15 d. Engraftment failure developed in one patient. The transplantation was followed by a 1-mo regimen of IL-2 (0.5 x 10(6) IU/m(2) daily) and GM-CSF, and 6 mo of IFN-alpha. One complete and one transient minor cytogenetic remission were observed. At 24 mo after transplantation, two patients had died of progressive disease and one of infection. Five patients had stable disease in the chronic phase. Autologous transplantation using IL-2-activated graft is feasible and the subsequent IL-2, GM-CSF, and IFN-alpha administration has acceptable toxicity. However, no benefits in comparison with conventional autologous transplantation for CML were identified in our study.