T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia.

The T-box transcription factor T-bet is known as a master regulator of T-cell response but its role in malignant B cells is not sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with genetic knockout of TBX21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity induced by inflammatory signals provided by the microenvironment, triggered T-bet expression which impacted on promoter proximal and distal chromatin co-accessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling, and a negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of CLL patients. Our study uncovers a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling which has implications for stratification and therapy of CLL patients. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in inflammatory signaling pathways in CLL.

Clonal evolution in chronic lymphocytic leukemia is scant in relapsed but accelerated in refractory cases after chemo(immune) therapy.

Clonal evolution is involved in the progression of chronic lymphocytic leukemia (CLL). In order to link evolutionary patterns to different disease courses, we performed a long-term longitudinal mutation profiling study of CLL patients. Tracking somatic mutations and their changes in allele frequency over time and assessing the underlying cancer cell fraction revealed highly distinct evolutionary patterns. Surprisingly, in long-term stable disease and in relapse after long-lasting clinical response to treatment, clonal shifts are minor. In contrast, in refractory disease major clonal shifts occur although there is little impact on leukemia cell counts. As this striking pattern in refractory cases is not linked to a strong contribution of known CLL driver genes, the evolution is mostly driven by treatment-induced selection of sub-clones, underlining the need for novel, non-genotoxic treatment regimens.

Prognostic and predictive role of gene mutations in chronic lymphocytic leukemia: results from the pivotal phase III study COMPLEMENT1.

Next generation sequencing studies in Chronic lymphocytic leukemia (CLL) have revealed novel genetic variants that have been associated with disease characteristics and outcome. The aim of this study was to evaluate the prognostic value of recurrent molecular abnormalities in patients with CLL. Therefore, we assessed their incidences and associations with other clinical and genetic markers in the prospective multicenter COMPLEMENT1 trial (treatment naive patients not eligible for intensive treatment randomized to chlorambucil (CHL) vs. ofatumumab-CHL (O-CHL)). Baseline samples were available from 383 patients (85.6%) representative of the total trial cohort. Mutations were analyzed by amplicon-based targeted next generation sequencing (tNGS). In 52.2% of patients we found at least one mutation and the incidence was highest in NOTCH1 (17.0%), followed by SF3B1 (14.1%), ATM (11.7%), TP53 (10.2%), POT1 (7.0%), RPS15 (4.4%), FBXW7 (3.4%), MYD88 (2.6%) and BIRC3 (2.3%). While most mutations lacked prognostic significance, TP53 (HR2.02,p<0.01), SF3B1 (HR1.66,p=0.01) and NOTCH1 (HR1.39,p=0.03) were associated with inferior PFS in univariate analysis. Multivariate analysis confirmed the independent prognostic role of TP53 for PFS (HR1.71,p=0.04) and OS (HR2.78,p=0.02) and of SF3B1 for PFS only (HR1.52,p=0.02). Notably, NOTCH1 mutation status separates patients with a strong and a weak benefit from ofatumumab addition to CHL (NOTCH1wt:HR0.50,p<0.01, NOTCH1mut:HR0.81,p=0.45). In summary, TP53 and SF3B1 were confirmed as independent prognostic and NOTCH1 as a predictive factor for reduced ofatumumab efficacy in a randomized chemo (immune)therapy CLL trial. These results validate NGS-based mutation analysis in a multicenter trial and provide a basis for expanding molecular testing in the prognostic workup of patients with CLL. ClinicalTrials.gov registration number: NCT00748189.

DNA methylation of chronic lymphocytic leukemia with differential response to chemotherapy.

Acquired resistance to chemotherapy is an important clinical problem and can also occur without detectable cytogenetic aberrations or gene mutations. Chronic lymphocytic leukemia (CLL) is molecularly well characterized and has been elemental for establishing central paradigms in oncology. This prompted us to check whether specific epigenetic changes at the level of DNA methylation might underlie development of treatment resistance. We used Illumina Infinium HumanMethylation450 BeadChips to obtain DNA methylation profiles of 71 CLL patients with differential responses. Thirty-six patients were categorized as relapsed/refractory after treatment with fludarabine or bendamustine and 21 of them had genetic aberrations of TP53. The other 35 patients were untreated at the time of sampling and 15 of them had genetic aberration of TP53. Although we could not correlate chemoresistance with epigenetic changes, the patients were comprehensively characterized regarding relevant prognostic and molecular markers (e.g. IGHV mutation status, chromosome aberrations, TP53 mutation status, clinical parameters), which makes our dataset a unique and valuable resource that can be used by researchers to test alternative hypotheses.

Oxidative stress as candidate therapeutic target to overcome microenvironmental protection of CLL.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6-8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

From Biology to Therapy: The CLL Success Story.

Chemoimmunotherapy has been the standard of care for patients with chronic lymphocytic leukemia (CLL) over the last decade. Advances in monoclonal antibody technology have resulted in the development of newer generations of anti-CD20 antibodies with improved therapeutic effectiveness. In parallel, our knowledge about the distinctive biological characteristics of CLL has progressively deepened and has revealed the importance of B-cell receptor (BCR) signaling and upregulated antiapoptotic proteins for survival and expansion of malignant cell clones. This knowledge provided the basis for development of novel targeted agents that revolutionized treatment of CLL. Ibrutinib and idelalisib inhibit the Bruton tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) delta, respectively, thus interfering with supportive signals coming from the microenvironment via the BCR. These drugs induce egress of CLL cells from secondary lymphoid organs and remarkably improve clinical outcomes, especially for patients with unmutated immunoglobulin heavy-chain genes or with p53 abnormalities that do not benefit from classical treatment schemes. Latest clinical trial results have established ibrutinib with or without anti-CD20 antibodies as the preferred first-line treatment for most CLL patients, which will reduce the use of chemoimmunotherapy in the imminent future. Further advances are achieved with venetoclax, a BH3-mimetic that specifically inhibits the antiapoptotic B-cell lymphoma 2 protein and thus causes rapid apoptosis of CLL cells, which translates into deep and prolonged clinical responses including high rates of minimal residual disease negativity. This review summarizes recent advances in the development of targeted CLL therapies, including new combination schemes, novel BTK and PI3K inhibitors, spleen tyrosine kinase inhibitors, immunomodulatory drugs, and cellular immunotherapy.

FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL.

NOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Control of chronic lymphocytic leukemia development by clonally-expanded CD8+ T-cells that undergo functional exhaustion in secondary lymphoid tissues.

Chronic lymphocytic leukemia (CLL) is associated with substantial alterations in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. Here, we utilized the Eµ-TCL1 mouse model of CLL as well as blood and lymph node samples of CLL patients to investigate the existence of anti-tumoral immune responses in CLL, and to characterize involved immune cell populations. Thereby, we identified an oligoclonal CD8+ effector T-cell population that expands along with CLL progression and controls disease development. We further show that a higher percentage of CD8+ effector T-cells produces IFNγ, and demonstrate that neutralization of IFNγ results in faster CLL progression in mice. Phenotypical and functional analyses of expanded CD8+ effector T-cells show significant differences in disease-affected tissues in mice, with cells in secondary lymphoid organs harboring hallmarks of activation-induced T-cell exhaustion. Notably, we further describe a respective population of exhausted CD8+ T-cells that specifically accumulate in lymph nodes, but not in peripheral blood of CLL patients. Collectively, these data emphasize the non-redundant role of CD8+ T-cells in suppressing CLL progression and highlight their dysfunction that can be exploited as target of immunotherapy in this malignancy.

Cytotoxicity of gypsogenic acid isolated from Gypsophila trichotoma.

BACKGROUND: Gypsophila trichotoma Wend. (Caryophyllaceae) is a medicinal plant which is protected in Bulgaria by the Biodiversity Law. Previous studies have showed the presence of triterpene saponins, sterols, flavonoids, triterpens, etc. OBJECTIVE: Gypsogenic acid, isolated from Gypsophila trichotoma roots, was evaluated for cytotoxic activity. MATERIALS AND METHODS: The structure of the compound was elucidated by spectral methods. The cell survival fraction was determined by the MTT dye reduction assay, performed with some modifications. RESULTS: Gypsogenic acid was tested in a panel of human tumor cell lines and was found to inhibit the proliferation of malignant cells. It was active against leukemic cells with lymphoid (SKW-3 and BV-173) or myeloid phenotype (HL-60, K-562, and LAMA-84), as well as against the EJ bladder carcinoma cell line. Bcr-Abl expressing myeloid cells (LAMA-84 and especially K-562) displayed lower sensitivity. HL-60/Dox cells were less sensitive to gypsogenic acid than the parent cell line, which shows that gypsogenic acid is probably a substrate of MRP-1.

Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines.

While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80µM (HH cells). The IC50 values of curcumin ranged from 12 to 24µM. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy.

A particular expression pattern of CD13 epitope 7H5 in chronic lymphocytic leukaemia--a possible new therapeutic target.

A total of 50 patients with chronic lymphocytic leukaemia (CLL), as well as the B-cell leukaemia cell lines MEC-1, JVM-3, and BV-173 were studied in order to assess the incidence of CD13/aminopeptidase N (APN) immunolabelling with a monoclonal antibody 7H5 compared to LeuM7 and to CD13 mRNA levels, and to correlate these data with the cytotoxic and apoptosis-induction activity of the natural phenolic APN inhibitor curcumin. CD13/APN was detected in a significant proportion of B-CLL patients (42/50, 84%), immunolabelled by 7H5 (42/50) ± LeuM7 (10/50). Molecular analysis for CD13 transcripts confirmed these data, resulting in a specific RT-PCR product in CD13 positive cases. Curcumin showed concentration-dependent cytoreductive efficacy and apoptosis-induction activity in all tested cell lines and primary cultures from CLL mononuclear cells. There was a clear tendency for a better response in CD13 positive cases. The incidence of CD13/APN in CLL suggests that the inhibition of APN/CD13 by curcumin may be an effective new molecular target for a more efficient therapy for these patients and warrants further investigations.

Interleukin-6, osteopontin and Raf/MEK/ERK signaling modulate the sensitivity of human myeloma cells to alkylphosphocholines.

Alkylphosphocholines are highly active against multiple myeloma (MM) cells in vitro and are devoid of myelotoxicity. Little is known about the determinants of MM cell responsiveness or resistance to these drugs. In this study we investigated the effects of disease-relevant cytokines, such as interleukin-6 (IL-6) and osteopontin (OPN), on the in vitro antimyeloma activity of erufosine and perifosine. The role of the Raf/MEK/ERK pathway was also studied. Exogenous IL-6 reduced the cytotoxicity of erufosine against OPM-2 cells and, to a smaller extent, against U-266 cells. This was accompanied by inhibition of apoptosis in OPM-2 cells. The efficacy of perifosine was similarly affected, but to a greater extent. IL-6 slightly enhanced the sensitivity of RPMI-8226 cells to erufosine, thus emphasizing the heterogeneity of MM. Induced overexpression of OPN isoforms made OPM-2 cells less sensitive to erufosine. In all cases of IL-6- or OPN-induced resistance, the effective concentrations of erufosine were still within the clinically achievable range. Like other alkylphosphocholines, erufosine enhanced Raf/MEK/ERK signaling in MM cells but in some cases this contributed to cytotoxicity.

Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is a potential antimyeloma drug devoid of myelotoxicity.

PURPOSE: Erufosine is an i.v. injectable alkylphosphocholine which is active against various haematological malignancies in vitro. In the present study, its effects on multiple myeloma (MM) cell lines and on murine and human hematopoietic progenitor cells (HPCs) were investigated. METHODS: The following MM cell lines were used: RPMI-8226, U-266 and OPM-2. The cytotoxicity of erufosine against these cell lines was determined by the MTT-dye reduction assay. Bcl-2, Bcl-X(L) and pAkt expression levels, activation of caspases, as well as cleavage of PARP, were studied by Western blotting. Migration was evaluated by a modified Boyden-chamber assay. The haematologic toxicity of erufosine was assessed using clonogenicity assays with normal HPCs of murine or human origin. RESULTS: Significant cytotoxic activity of erufosine against the MM cell lines was found. Comparison of the characteristics of erufosine-induced cell death in the three cell lines revealed a complex mode of action with apoptotic mechanisms prevailing in OPM-2 cells and non-apoptotic mechanisms prevailing in U-266 cells. The sensitivity of the MM cell lines to erufosine-induced apoptosis correlated inversely with the Bcl-X(L) expression level. Erufosine participated in synergistic interactions with various drugs. Furthermore, it showed potent migration-inhibiting activity in RPMI-8226 cells. Erufosine was not toxic to normal HPCs of murine or human origin and even stimulated progenitors from human umbilical cord blood to form granulocyte/macrophage colonies. Moreover, erufosine ameliorated the toxicity of bendamustine to murine HPCs. CONCLUSIONS: Overall, the data presented reveal that erufosine could have potential as an antimyeloma drug and deserves further development.

Erucylphospho-N,N,N-trimethylpropylammonium shows substantial cytotoxicity in multiple myeloma cells.

Multiple myeloma (MM) is a frequent hematological malignancy that is incurable despite recent developments, such as proteasome and angiogenic inhibitors. Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is an i.v. injectable alkylphosphocholine with antineoplastic activity based on an unusual mode of action and is currently undergoing clinical trials in leukemia patients. The aim of this investigation was to evaluate the efficacy of erufosine in MM cells and to study the modulation of cell-death pathways. The cytotoxicity of erufosine against three MM cell lines (RPMI-8226, U-266, and OPM-2) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dye reduction assay. All MM cell lines responded to erufosine, RPMI-8226 cells being most and U-266 being least sensitive. The respective IC(50) values were 3.2 and 16.2 micromol/L. Various cell-death characteristics were studied in response to erufosine, such as morphological changes, oligonucleosomal DNA fragmentation, caspase activation, and poly (ADP)-ribose polymerase cleavage. Erufosine was found to cause cell shrinkage, chromatin condensation, and caspase-8 and -3 activation. Taken together, our data indicate that erufosine is a potential antimyeloma drug eliciting specific features of apoptotic cell death in vitro.

The expression level of the tumor suppressor retinoblastoma protein (Rb) influences the antileukemic efficacy of erucylphospho-N,N,N-trimethylpropylammonium (ErPC3).

The alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) is a promising new drug for treating various types of cancer. Its mechanism of action is not yet fully understood but is related to the Rb tumor suppressor protein. In the present study, we investigated the role of decreased Rb expression levels for the antileukemic efficacy of ErPC3 in BV-173 and K-562 CML-derived cell lines. We used antisense technique to knock down Rb levels in the two cell lines in addition to ErPC3 treatment. Cells with reduced Rb expression showed a diminished sensitivity to ErPC3 exposure, as determined by MTT (BV-173 and K-562) and clonogenicity assays (K-562 only), if concentrations below the IC50 were used. The feasibility of Rb knockdown varied between BV-173 and K-562 cells, with the former being distinctly more sensitive than the latter. We conclude that sufficient Rb levels are important for the cytotoxic and anticlonogenic effects of ErPC3 at levels below the IC(50), but that higher concentrations of ErPC3 are less dependent on Rb status.