Optimizing Drug Combinations for T-PLL: Restoring DNA Damage and P53-Mediated Apoptotic Responses.

T-prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm associated with marked chemotherapy resistance and continued poor clinical outcomes. Current treatments, i.e. the CD52-antibody alemtuzumab, offer transient responses, with relapses being almost inevitable without consolidating allogeneic transplantation. Recent more detailed concepts of T-PLL's pathobiology fostered the identification of actionable vulnerabilities: (i) altered epigenetics, (ii) defective DNA damage responses, (iii) aberrant cell-cycle regulation, and (iv) deregulated pro-survival pathways, including TCR and JAK/STAT signaling. To further develop related pre-clinical therapeutic concepts, we studied inhibitors of (H)DACs, BCL2, CDK, MDM2, and clas-sical cytostatics, utilizing (a) single-agent and combinatorial compound testing in 20 well-characterized and molecularly-profiled primary T-PLL (validated by additional 42 cases), and (b) 2 independent murine models (syngeneic transplants and patient-derived xenografts). Overall, the most efficient/selective single-agents and combinations (in vitro and in mice) in-cluded Cladribine, Romidepsin ((H)DAC), Venetoclax (BCL2), and/or Idasanutlin (MDM2). Cladribine sensitivity correlated with expression of its target RRM2. T-PLL cells revealed low overall apoptotic priming with heterogeneous dependencies on BCL2 proteins. In additional 38 T-cell leukemia/lymphoma lines, TP53 mutations were associated with resistance towards MDM2 inhibitors. P53 of T-PLL cells, predominantly in wild-type configuration, was amenable to MDM2 inhibition, which increased its MDM2-unbound fraction. This facilitated P53 activa-tion and down-stream signals (including enhanced accessibility of target-gene chromatin re-gions), in particular synergy with insults by Cladribine. Our data emphasize the therapeutic potential of pharmacologic strategies to reinstate P53-mediated apoptotic responses. The identified efficacies and their synergies provide an informative background on compound and patient selection for trial designs in T-PLL.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity.

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Transcriptional activation of hTERT, the human telomerase reverse transcriptase, by nuclear factor of activated T cells.

Telomerase is essential for telomere maintenance, and its activation is thought to be a critical step in cellular immortalization and tumorigenesis. Human telomerase reverse transcriptase (hTERT) is a major component of telomerase activity. We show here that hTERT is expressed soon after lymphocyte activation and that its expression is inhibited by rapamycin, wortmannin, and FK506, which was the most potent inhibitor. These results suggest a potential role for the transcription factor nuclear factor of activated T cells (NFAT) in the regulation of hTERT expression. Five putative NFAT-binding sites were identified in the hTERT promoter. In luciferase assays, the hTERT promoter was activated by overexpressed NFAT1. Moreover, serial deletions revealed that the promoter activation was mainly due to a -40 NFAT1-binding site flanked by two SP1-binding sites. Mutation of the -40 NFAT-binding site caused a 53% reduction in the transcriptional activity of hTERT promoter. Simultaneous mutations of the -40 NFAT-responsive element together with one or both SP1-binding sites led to a more dramatic decrease in luciferase activity than single mutations, suggesting a functional synergy between NFAT1 and SP1 in hTERT transcriptional regulation. NFAT1 overexpression in MCF7 and Jurkat cell lines induced an increase in endogenous hTERT mRNA expression. Inversely, its down-regulation was induced by NFAT1 silencing. Furthermore, chromatin immunoprecipitation assay demonstrated that NFAT1 directly binds to two sites (-40 and -775) in the endogenous hTERT promoter. Thus, we show for the first time the direct involvement of NFAT1 in the transcriptional regulation of hTERT.

Two new miR-16 targets: caprin-1 and HMGA1, proteins implicated in cell proliferation.

BACKGROUND INFORMATION: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16. RESULTS: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. CONCLUSIONS: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.

Development of a Model for Chemical Screening Based on Collateral Sensitivity to Target BTK C481S Mutant.

Targeted therapies have improved the outcome of cancer, but their efficacy is intrinsically limited by the emergence of subclones with a mutation in the gene encoding the target protein. A few examples of collateral sensitivity have demonstrated that the conformational changes induced by these mutations can create unexpected sensitivity to other kinase inhibitors, but whether this concept can be generalized is unknown. Here is described the development of a model to screen a library of kinase inhibitors for collateral sensitivity drugs active on the Bruton Tyrosine Kinase (BTK) protein with the ibrutinib resistance mutation C481S. First, we demonstrate that overexpression of the constitutively active mutant of BTK harboring the E41K mutation in Ba/F3 cells creates an oncogenic addiction to BTK. Then, we have exploited this phenotype to perform a screen of a kinase inhibitor library on cells with or without the ibrutinib resistance mutation. The BTK inhibitors showed the expected sensitivity profile, but none of the drugs tested had a specific activity against the C481S mutant of BTK, suggesting that extending the collateral sensitivity paradigm to all kinases targeted by cancer therapy might not be trivial.

Does p16ink4a expression increase with the number of cell doublings in normal and malignant lymphocytes?

p16(ink4a) is known to be a major inhibitor of cyclin-dependent kinases of G1-phase. Its accumulation is associated with replicative senescence. We analyzed to what extent the number of cell doublings may participate to p16(ink4a) expression in normal and malignant lymphocytes. p16(ink4a) expression, not found in normal quiescent B or T-lymphocytes, was observed after stimulation of B-lymphocytes (72 h) and T-lymphocytes (2 weeks) before the occurrence of replicative senescence markers such as senescence-associated-beta-galactosidase activity. Afterwards, in lymphocyte long-term cultures, the increase in p16(ink4a) followed the expression of features of cell ageing. In acute lymphoblastic leukemia, the analysis of the individual differences between peripheral blood and blood compartments (34 cases) showed a decrease in cell proliferation (p<0.005), in telomerase activity (p<0.0005), and in hTERT expression (p<0.04), associated with an increase of p16(ink4a) (p<0.035) in blood leukemic cells. These results support the hypothesis that (i) an increase in p16(ink4a) expression in normal lymphocytes is linked, in part, to the number of cell doublings before the occurrence of replicative senescence and (ii) this process is maintained in leukemic cell populations of numerous patients.

Rapamycin safeguards lymphocytes from DNA damage accumulation in vivo.

Several studies reported the benefits of switching from anticalcineurins to mTOR inhibitors to avoid cancer occurrence after organ transplantation. The purpose of our study was to determine in vivo biological markers to explain these benefits. Cellular changes related to cellular senescence and DNA damage were analyzed in peripheral blood lymphocytes. Thirty-five kidney transplanted patients receiving anticalcineurins were investigated: 17 patients were proposed to switch to rapamycin and 18 patients with similar age and transplantation duration, continued anticalcineurins. Rapamycin effects were studied one year after the switch. Thirteen healthy volunteers and 18 hemodialyzed patients were evaluated as control. Compared with the healthy group, hemodialyzed and transplanted patients exhibited a significant decrease in telomere length, an increase in p16(INK4A) mRNA expression and in lymphocytes with 53BP1 foci. A destabilization of the shelterin complexes was suggested by a significant TIN2 mRNA decrease in transplanted patients compared with controls and a significant increase in TRF1, TRF2 and POT1 expression in switch-proposed patients compared with the non-switched subgroup. Rapamycin treatment resulted in a significant decrease in DNA damage and a slight TIN2 increase. In vitro experiments strengthened in vivo results showing that rapamycin but not FK506 induced a significant DNA damage decrease and TIN2 expression increase compared with controls. The roles of rapamycin in the decrease in DNA damage in vivo and the rescue of shelterin gene expression are demonstrated for the first time. These data provide new insights into understanding of how rapamycin may overcome genomic injuries.

The ibrutinib B-cell proliferation inhibition is potentiated in vitro by dexamethasone: Application to chronic lymphocytic leukemia.

New B-cell receptor-targeted therapies such as ibrutinib, a Bruton tyrosine kinase inhibitor, are now proposed for lymphoid pathologies. The putative benefits of its combination with glucocorticoids were evaluated here. We compared the effects of dexamethasone (DXM), ibrutinib and their in vitro combination on proliferation and metabolic stress markers in stimulated normal B-lymphocytes and in malignant lymphocytes from chronic lymphocytic leukemia (CLL) patients. In both cellular models, cell cycle progression was globally inhibited by DXM and/or ibrutinib. This inhibition was significantly amplified by DXM addition to ibrutinib and was related to a significant decrease in the expression of the cell cycle regulatory proteins CDK4 and cyclin E. Apoptosis increased especially with DXM/ibrutinib combination and was associated with a significant decrease in Mcl-1 expression. Treatment effects on metabolic stress were evaluated by DNA damage recognition after 53BP1 foci labeling. The percentage of cells with more than five 53BP1 foci decreased significantly with ibrutinib in normal and CLL lymphocytes. This decrease was strongly reinforced, in CLL, by DXM addition. Our data indicated that, in vitro, DXM potentiated antiproliferative effects of ibrutinib and decreased DNA damage in lymphoid B-cells. Thus their combination may be proposed for CLL treatment.

The p16(INK4A)/pRb pathway and telomerase activity define a subgroup of Ph+ adult Acute Lymphoblastic Leukemia associated with inferior outcome.

Adult Acute Lymphoblastic Leukemia (ALL) therapies have been improved by pediatric-like approaches. However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The p16(INK4A)/CDK4-6/pRb pathway and telomerase activity, which are implicated in cell activation and aging, were analyzed to identify new prognostic markers. Proteins of the p16(INK4A)/CDK4-6/pRb pathway and telomerase activity were analyzed in 123 adult B-cell precursor (BCP) ALL cases included in the GRAALL/GRAAPH trials. We found a significantly increased expression of p16(INK4A) in BCP-ALLs with MLL rearrangement. Telomerase activity was significantly lower in Philadelphia chromosome-negative/IKAROS-deleted (BCR-ABL1(-)/IKAROS(del)) cases compared to Philadelphia chromosome-positive (BCR-ABL1+) BCP-ALLs. In BCR-ABL1+ ALLs, high CDK4 expression, phosphorylated pRb (p-pRb) and telomerase activity were significantly associated with a shorter disease-free survival (DFS) and event-free survival (EFS). Enhanced p16(INK4A) expression was only related to a significantly shorter DFS. In vitro analyses of normal stimulated lymphocytes after short- and long-term cultures demonstrated that the observed protein variations of poor prognosis in BCR-ABL1+ ALLs may be related to cell activation but not to cell aging. For these patients, our findings argue for the development of therapeutic strategies including the addition of new lymphocyte activation inhibitors to current treatments.

Transcriptional regulation of the human telomerase reverse transcriptase: new insights.

hTERT and NFAT were thought until recently to belong to separate metabolic compartments. The involvement of NFAT in the induction of hTERT transcription suggested by hTERT expression variations during lymphocyte stimulation and immunosuppressive treatments explains the link between hTERT expression and cell stimulation and offers new insights for therapeutic developments.

Indirubin derivatives inhibit malignant lymphoid cell proliferation.

Indirubin-3'-monoxime (IO) is a derivative of indirubin, an active compound of a traditional Chinese medicinal recipe used to treat various inflammatory and malignant diseases. The main in vitro targets of IO (i.e. cyclin dependent kinases, glycogen synthase kinase-3beta, Stat 3 and Aryl hydrocarbon receptor) are regulators of lymphocyte activation. We investigated the interest of IO and its derivative 6-bromo-indirubin-3'oxime (6BIO) for inhibiting the growth of malignant lymphoid cells. IO (1-20 microM) induced cell cycle inhibition and cell death in malignant B- (IM9, Reh6) and T- (Jurkat, CEM-T) lymphoid cell lines depending to cell type, doses, and duration of treatment. IO and 6BIO (10 microM) treatment for 24 and 48 h were compared: 6BIO treatment resulted in a stronger cytotoxicity and more profound inhibition of cell proliferation. Taken together, these results showed that IO and, moreover, its derivative 6BIO may be potent antiproliferative agents in malignant lymphoid cells.

Telomere uncapping during in vitro T-lymphocyte senescence.

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in gamma-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16(ink4a) upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.