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Simple Summary The tyrosine kinase inhibitor (TKI) imatinib was the first targeted therapy to show clinical efficacy against chronic myeloid leukemia (CML) through inhibition of the breakpoint cluster region–Abelson murine leukemia viral oncogene homolog (BCR-ABL), which is responsible for the disease. Two other generations of TKIs have succeeded imatinib, offering additional therapeutic solutions for a growing number of patients with imatinib-resistant CML. However, these clinical approaches although very effective, generate many unwanted side effects because of their daily administration. Attempts to stop TKI when the disease is no longer detectable at the molecular level, unfortunately result in relapses in more than half of cases. This highlights the presence of undetectable leukemia cells, recognized as leukemic stem cells (LSCs) that are TKI insensitive. It therefore appears necessary to identify new biochemical pathways in LSCs, the targeting of which would make re-sensitization to TKIs possible. The results presented here demonstrate that targeting the protein kinase Cδ (PKCδ) pathway represents a valid alternative for LSC elimination. Abstract Numerous combinations of signaling pathway blockades in association with tyrosine kinase inhibitor (TKI) treatment have been proposed for eradicating leukemic stem cells (LSCs) in chronic myeloid leukemia (CML), but none are currently clinically available. Because targeting protein kinase Cδ (PKCδ) was demonstrated to eliminate cancer stem cells (CSCs) in solid tumors, we evaluated the efficacy of PKCδ inhibition in combination with TKIs for CML cells. We observed that inhibition of PKCδ by a pharmacological inhibitor, by gene silencing, or by using K562 CML cells expressing dominant-negative (DN) or constitutively active (CA) PKCδ isoforms clearly points to PKCδ as a regulator of the expression of the stemness regulator BMI1. As a consequence, inhibition of PKCδ impaired clonogenicity and cell proliferation for leukemic cells. PKCδ targeting in K562 and LAMA-84 CML cell lines clearly enhanced the apoptotic response triggered by any TKI. A strong synergism was observed for apoptosis induction through an increase in caspase-9 and caspase-3 activation and significantly decreased expression of the Bcl-xL Bcl-2 family member. Inhibition of PKCδ did not modify BCR-ABL phosphorylation but acted downstream of the oncogene by downregulating BMI1 expression, decreasing clonogenicity. PKCδ inhibition interfered with the clonogenicity of primary CML CD34+ and BCR-ABL-transduced healthy CD34+ cells as efficiently as any TKI while it did not affect differentiation of healthy CD34+ cells. LTC-IC experiments pinpointed that PKCδ inhibition strongly decreased the progenitors/LSCs frequency. All together, these results demonstrate that targeting of PKCδ in combination with a conventional TKI could be a new therapeutic opportunity to affect for CML cells.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

International audience; Iron is an essential nutrient, acting as a catalyst for metabolic reactions that are fundamental to cell survival and proliferation. Iron complexed to transferrin is delivered to the metabolism after endocytosis via the CD71 surface receptor. We found that transformed cells from a murine PTEN-deficient T-cell lymphoma model and from T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LL) cell lines overexpress CD71. As a consequence, the cells developed an addiction toward iron whose chelation by deferoxamine (DFO) dramatically affected their survival to induce apoptosis. Interestingly, DFO displayed synergistic activity with three ALL-specific drugs: dexamethasone, doxorubicin, and L-asparaginase. DFO appeared to act through a reactive oxygen species-dependent DNA damage response and potentiated the action of an inhibitor of the PARP pathway of DNA repair. Our results demonstrate that targeting iron metabolism could be an interesting adjuvant therapy for acute lymphoblastic leukemia.

We show here that the antidiabetic agents metformin and phenformin and the AMPK activator AICAR exert strong anti-tumoural effects on tPTEN−/− lymphoma cells and on human T-ALL cell lines and primary samples. The compounds act by inhibiting tumour metabolism and proliferation and by inducing apoptosis in parallel with an activation of AMPK and an inhibition of constitutive mTOR. In tPTEN−/− cells, the drugs potentiated the anti-leukaemic effects of dexamethasone, and metformin and phenformin synergised with 2-deoxyglucose (2DG) to impair tumour cell survival. In vivo, metformin and AICAR strongly decreased the growth of luciferase-expressing tPTEN−/− cells xenografted in Nude mice, demonstrating that metabolism targeting could be a potent adjuvant strategy for lymphoma/leukaemia treatment.

Testicular germ cell cancers are the most common solid malignancies in young men, but their pathogenesis remains undetermined although some epidemiological data have implicated both environmental and genetic factors. Glial cell-derived neurotrophic factor (GDNF) is secreted by Sertoli cells, and promotes germ stem cell proliferation by activating RET, a tyrosine kinase receptor. Over-expression of GDNF in adult transgenic mice induces the development of testicular tumours that mimic human seminoma, the most frequent testicular germ cell tumour. Activating mutations of RET were previously reported in several types of cancer, including thyroid, pituitary, adrenal and melanoma cancer. Both mouse experimental model and clinical studies suggested that mutations or selective polymorphisms of RET might be associated with human seminoma. To verify this hypothesis, we conducted this study in a French University Hospital and carried out an association study using tissue samples from 66 paraffin-embedded seminoma tumours. The most frequently mutated exons of the RET proto-oncogene were sequenced to identify mutations or selective polymorphisms. No somatic mutations were identified. The polymorphic variants frequencies did not differ from those in a control Caucasian population. Human classical seminoma that occurs in young men does not appear to be linked with mutations or relevant polymorphisms of RET.

Summary Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (αFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2γ and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.

Background Fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, many of these xenoestrogens have only a weak affinity for the classical estrogen receptors (ERs,) which is 1,000-fold less potent than the affinity of 17β-estradiol (E2). Thus, several mechanisms have been suggested to explain how they could affect male germ cell proliferation at low environmental relevant concentrations. Objectives In this study we aimed to explore the possible promoting effect of bisphenol A (BPA) on human testicular seminoma cells. BPA is a well-recognized estrogenic endocrine disruptor used as a monomer to manufacture poly carbonate plastic and released from resin-lined food or beverage cans or from dental sealants. Methods and results BPA at very low concentrations (10−9 to 10−12 M) similar to those found in human fluids stimulated JKT-1 cell proliferation in vitro. BPA activated both cAMP-dependent protein kinase and cGMP-dependent protein kinase pathways and triggered a rapid (15 min) phosphorylation of the transcription factor cAMP response-element–binding protein (CREB) and the cell cycle regulator retinoblastoma protein (Rb). This nongenomic activation did not involve classical ERs because it could not be reversed by ICI 182780 (an ER antagonist) or reproduced either by E2 or by diethylstilbestrol (a potent synthetic estrogen), which instead triggered a suppressive effect. This activation was reproduced only by E2 coupled to bovine serum albumin (BSA), which is unable to enter the cell. As with E2-BSA, BPA promoted JKT-1 cell proliferation through a G-protein–coupled nonclassical membrane ER (GPCR) involving a Gαs and a Gαi/Gαq subunit, as shown by the reversible effect observed by the corresponding inhibitors NF449 and pertussis toxin. Conclusion This GPCR-mediated nongenomic action represents—in addition to the classical ER-mediated effect—a new basis for evaluating xenoestrogens such as BPA that, at low doses and with a high affinity for this GPCR, could interfere with the developmental programming of fetal germ cell proliferation and/or differentiation when they cross the placenta.

Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LICs), detectable by their capacity to self-renew and generate non-LICs in vivo. The xenograft assay captures functional properties of LICs that have clinical prognostic value, but the long duration of xenotransplantation has hampered its implementation in the clinic. Here, we describe an ex vivo co-culture system in which the frequency of leukemic long-term culture-initiating cells (L-LTC-ICs) can be used as a reliable read-out of LIC activity over one week. We analyzed 92 AML patients classified as having a poor, intermediate, or favorable prognosis at time of diagnosis. Ex vivo limiting dilution analyses demonstrated that the frequency of L-LTC-ICs was 5-7 times higher in intermediate and poor prognosis samples compared with favorable samples after five weeks of co-culture with MS-5 stromal cells. To simplify and shorten this test, we defined a fluorescence dilution factor (FDF) as the ratio of the median fluorescence intensity, based on CFSE staining, before and after one week of co-culture. L-LTC-IC proliferation, monitored over one week, was strongly correlated to L-LTC-IC frequency. A higher FDF was evident in poor prognosis AMLs or in samples capable of engrafting NSG mice compared with favorable prognosis AMLs or non-engrafters. Notably, the FDF could classify intermediate prognosis patients with normal karyotype into two groups differing in overall survival. This non-genetic and non-in vivo approach represents a new clinically relevant tool for rapidly and reliably predicting AML patient outcome.

Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

Histone lysine methyl transferase 2 (EZH2) inhibitor GSK126 and a novel deuterated internal standard GSK126-d7 were chemically prepared. We performed in vitro experiments using the prepared GSK126 to: i) confirm in vitro EZH2 inhibitory activity; ii) conduct Sprague-Dawley (SD) rat liver microsomal incubations and identified Phase I metabolites; iii) determine whether or not GSK126 was an Organic Anion Transporter (OAT) substrate; and, iv) determine oral bioavailability by conducting oral and orbital sinus dosing (OSD) experiments and determining blood concentration versus time profiles. GSK126 was shown to decrease the expression of H3K27Me3 protein in medulloblastoma D283 cells and was able to decrease cell viability in KO99L cells, a novel T cell lymphoma cell line. Three in vitro hepatic Phase I mono-oxidative metabolites (L-M1, L-M2 and L-M3) were observed and also detected in rat liver and urine samples from the in vivo studies. GSK126 was found to be an OAT1 and OAT2 substrate, but not an OAT3 or OAT4 substrate. Our Pharmacokinetic (PK) results indicate: 1) GSK126 has very poor oral bioavailability (< 2%); 2) co-administration of probenecid, a prototypical OAT inhibitor, did not significantly alter observed PK; and 3) tissue distribution studies demonstrate that GSK126 predominately distributes to the liver and kidneys after an OSD.

There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.

The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute lymphoblastic leukemia (T-ALL)/lymphoma (T-LL) cells. We show here that a potent and LAT1 selective inhibitor (JPH203) decreased leukemic cell viability and proliferation, and induced transient autophagy followed by apoptosis. JPH203 could also alter the in vivo growth of luciferase-expressing-tPTEN-/- cells xenografted into nude mice. In contrast, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes. JPH203 interfered with constitutive activation of mTORC1 and Akt, decreased expression of c-myc and triggered an unfolded protein response mediated by the C/EBP homologous protein (CHOP) transcription factor associated with cell death. A JPH203-resistant tPTEN-/-clone appeared CHOP induction deficient. We also demonstrate that targeting LAT1 may be an efficient broad spectrum adjuvant approach to treat deadly T-cell malignancies as the molecule synergized with rapamycin, dexamethasone, doxorubicin, velcade and l-asparaginase to alter leukemic cell viability.

Macroautophagy (hereafter referred to as autophagy) is the major degradative pathway of long-lived proteins and organelles that fulfils key functions in cell survival, tissue remodeling and tumor suppression. Consistently, alterations in autophagy have been involved in a growing list of pathologies including toxic injury, infections, neurodegeneration, myopathies and cancers. Although critical, the molecular mechanisms that control autophagy remain largely unknown. We have recently exploited the disruption of autophagy by environmental carcinogens as a powerful model to uncover the underlying signaling pathways. Our work published in Cancer Research revealed that the sustained activation of the MAPK ERK pathway by the carcinogen Lindane or the MEK1(+) oncogene alters autophagy selectively at the maturation step resulting in the accumulation of large defective autolysosomes. Consistent with our findings, a similar defect is observed with other common xenobiotics such as dichlorodiphenyltrichloroethane and biphenol A that specifically activate ERK. Conversely, Pentachlorophenol that activates both ERK and p38, fails to induce autophagic vacuolation. In addition, evidence is provided that abrogation of p38 by SB203580 is sufficient to interfere with the normal autophagic maturation step. Altogether, these findings underscore the critical role played by MAPK ERK and p38 in the tight control of the autophagy process at the maturation step.

The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN−/− mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL.

Clinical and experimental studies have suggested that estrogens, the archetype of female hormones, participate in the control of male germ cell proliferation and that fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, the underlying mechanisms remain to be elucidated. 17β-Estradiol (E2) conjugated to BSA was able to stimulate human testicular seminoma cell proliferation by triggering a rapid, nongenomic, membrane-mediated activation of ERK1/2 and cAMP-dependent protein kinase A (PKA). Both ERK1/2 and PKA participated in this promoting effect. This activation was associated with phosphorylation of the transcription factor cAMP response element-binding protein and the nuclear factor retinoblastoma protein. Enhanced proliferation together with ERK activation could be reversed by pertussis toxin, a G protein inhibitor. Estrogen receptors (ERs) in JKT-1 were characterized by immunofluorescence, subcellular fractioning, and Western blot. JKT-1 cells did not express ERα but ERβ, which localized to the mitochondria and the nucleus but not to the membrane. Moreover, neither ICI-182,780, a classical ER antagonist, nor tamoxifen, a selective ER modulator, could reverse the 17β-estradiol-BSA-induced promoting effect. Estrogens contribute to human testicular germ cell cancer proliferation by rapid activation of ERK1/2 and PKA through a membrane nonclassical ER. This nongenomic effect represents a new basis for understanding the estrogenic control of spermatogenesis and evaluating the role of fetal exposure to xenoestrogens during malignant transformation of testicular germ stem cells.