Methods for determining the metabolic status of lymphomas

Abstract

Provided is an in vitro method for determining the metabolic status of a lymphoma comprising a step of determining the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in lymphoma cells, wherein a low level of GAPDH expression is indicative of oxidative phosphorylation (OXPHOS) status. Also provided is an in vitro method for predicting the responsiveness of a patient afflicted with a lymphoma to a treatment with a metabolic inhibitor selected from the group consisting of mitochondrial metabolic inhibitors and glutamine metabolism inhibitors comprising a step of determining the level of GAPDH expression in lymphoma cells obtained from said patient, wherein a low level of GAPDH expression is predictive of a response to a treatment with a metabolic inhibitor.

Claims

1. A method for treating a lymphoma patient in need thereof with a mitochondrial metabolic inhibitor and/or a glutamine metabolism inhibitor, comprising: a) determining a level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in lymphoma cells obtained from said patient, b) identifying the patient as a responder when the level of GAPDH expression is lower than a reference value obtained from healthy control subjects; and c) treating the patient identified in b) as a responder with a mitochondrial metabolic inhibitor and/or a glutamine metabolism inhibitor.

2. A method for treating a patient in need thereof with an immune checkpoint inhibitor, comprising: a) determining a level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in lymphoma cells obtained from the patient, b) identifying the patient as a responder when the level of GAPDH expression is lower than a reference value obtained from healthy control subjects; and c) treating the patient identified in b) as a responder with an immune checkpoint inhibitor.

3. The method according to claim 1, wherein the patient is afflicted with a non-Hodgkin's B cell lymphoma.

4. The method according to claim 3, wherein the non-Hodgkin's B cell lymphoma is diffuse large B-cell lymphoma (DLBCL).

5. The method according to claim 1, wherein the mitochondrial metabolic inhibitor is a mitochondrial complex I inhibitor.

6. The method according to claim 5, wherein the mitochondrial complex I inhibitor is selected from the group consisting of biguanides, rotenoids, piericidins, capsaicins, pyridinium-type inhibitors and thiazolidinediones.

7. The method according to claim 5, wherein the mitochondrial complex I inhibitor is a biguanide selected from the group consisting of metformin, phenformin and buformin.

8. The method according to claim 1, wherein the glutamine metabolism inhibitor is L-asparaginase.

9. The method according to claim 2, wherein the immune checkpoint inhibitor is selected from the group consisting of an antibody, synthetic sequence peptides, native sequence peptides, small molecules which bind to immune checkpoint proteins and their ligands, and aptamers which bind to immune checkpoint proteins and their ligands.

Description

FIGURES

(1) FIG. 1: Mouse primary E-Myc lymphomas express different levels of GAPDH. A. The relative levels of gapdh (left) and ldh-a (right) mRNA were determined by quantitative PCR in E-Myc-gapdh.sup.high clones (n=4 independent clones) and in E-Myc-gapdh.sup.low clones (n=5 independent clones). B. Quantification of GAPDH, HK2, ENO1, PKM2, PDK1 expression levels relative to Erk2 expression in E-Myc-gapdh.sup.high (n=4) and E-Myc-gapdh.sup.low (n=5) lymphomas (GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HK2: hexokinase 2; ENO1: enolase1; PKM2: pyruvate kinase M2; PDK1: pyruvate dehydrogenase kinase 1). The presented data are the means of 3 independent experiments expressed as meanSD. *p<0.05, ***p<0.001

(2) FIG. 2: E-Myc-gapdh.sup.low clones have a mitochondrial OXPHOS phenotype compared to E-Myc-gapdh.sup.high clones. Mitochondrial and glycolytic ATP production were measured in E-Myc-gapdh.sup.low (n=5 independent clones) and E-Myc-gapdh.sup.high (n=4 independent clones) clones as a percentage of total ATP production by the cells. The presented data are the means of 3 independent experiments.

(3) FIG. 3: Compared to E-Myc-gapdh.sup.high, E-Myc-gapdh.sup.low lymphomas are more sensitive to the mitochondrial complex I inhibitor, Phenformin and to glutamine/asparagine hydrolysis. A. EMyc-gapdh.sup.high (n=3 independent clones) and E-Myc-gapdh.sup.low (n=3 independent clones) cells were seeded in the presence or absence (Ctl) of phenformin (Phen, 200 M) for 24 hours. Cell death was determined by DAPI staining and analyzed by FACS. B. The proliferation index was measured in 2 independent E-Myc-gapdh.sup.low (clones #E and #G, upper panel) and in 2 independent E-Myc-gapdh.sup.high (clones #B and C, lower panel). C. E-Myc-gapdh.sup.high (n=3 independent clones) and E-Myc-gapdh.sup.low (n=3 independent clones) cells were seeded in the presence or absence (Ctl) of L-asparaginase (L-asp 0.3 UI/ml; 1.0 UI/ml) for 24 hours. Cell death was determined by DAPI staining and analyzed by flow cytometry.

(4) The presented data are the means of at least 3 independent experiments expressed as meanSD. **p<0.01, ***p<0.005.

(5) FIG. 4: Specific silencing of GAPDH sensitizes E-Myc-gapdh.sup.high lymphomas to phenformin and L-asparaginase. A. E-Myc-gapdh.sup.high cells silenced (shgapdh) or not (shctl) for gapdh were seeded in the presence or absence of 100 M of phenformin for 24 and 48 hours. Cells were then labeled with DAPI (Molecular Probes; 0.5 g/mL) and analyzed immediately by flow cytometry. B. The proliferation index was measured after 24 and 48 hours of phenformin treatment (50 M) in E-Myc-gapdh.sup.high cells silenced (shgapdh) or not (shctl) for gapdh cells. C. E-Myc-gapdh.sup.high cells silenced (shgapdh) or not (shctl) for gapdh were seeded in the presence or absence of L-asparaginase (0.3 UI/ml) for 24 hours. Cells were then labeled with DAPI (Molecular Probes; 0.5 g/mL) and analyzed by flow cytometry.

(6) FIG. 5: Phenformin reduced lymphomas progression in E-Myc-gapdh.sup.low-bearing mice. A. Kaplan-Meier curves of progression-free survival of syngenic C57BL/6 mice intravenously injected with primary E-Myc-gapdh.sup.low cells and treated or not (Ctl) with 200 mg/kg/day of Phenformin for 12 days (n=10 mice per group). B. C. Upon sacrifice of the animals, two axillary lymph nodes (B.) and the spleen (C.) were weighted (n=10 per group).

(7) FIG. 6: Follicular lymphomas relying on OxPhos metabolism for energy production express low levels of gapdh mRNA. A. Mitochondrial (OxPhos) and glycolytic ATP were measured as a percentage of total cellular ATP produce by the tumor cells, immediately after cell dissociation of freshly harvested biopsies diagnosed as follicular lymphomas (displaying at least 70% of B cells). B. Real time qPCR analysis of gapdh mRNA levels from follicular lymphomas relying on glycolysis (n=6) or OxPhos (n=5) for ATP production. Transcript levels were determined relative to cyclophilin-A (ppia) mRNA levels. gapdh mRNA levels expressed in tumor biopsies were then normalized to that expressed in normal B cells.

(8) TABLE-US-00001 TABLE 1 Effect of combined mitochondrial inhibitors on the therapeutic response of R-CHOP-refractory DLBCL expressing low or high level of GAPDH at diagnosis (clinical trial KTMR). Nine R-CHOP refractory Myc+ -DLBCL patients were enrolled in a new clinical protocol which combined L-asparaginase, Torisel (mTOR inhibitor), Metformine and Rituximab. Out of nine, four patients experienced toxicity and were removed from the study. Immunohistochemical staining of GAPDH was performed on tumor sections obtained from biopsies at diagnosis to determine whether GAPDH could be used as a predictive marker of KTMR therapeutic response. L-asparaginase + Torisel + Metformine + Rituximab GAPDH expression (IHC) - Complete biopsies at diagnosis Progression Partial Response Response GAPDH high 0 1 0 % 0 100 0 GAPDH low 1 0 3 % 25 0 75 4 patients were out of the study for toxicity reasons

(9) Effect of combined mitochondrial inhibitors on the therapeutic response of R-CHOP-refractory DLBCL displaying low or high GAPDH expression at diagnosis (n=9).

Example

(10) Material & Methods

(11) Cell Culture:

(12) Mouse primary E-Myc clones (B lymphoma cells) obtained from lymphomas of different C57BL/6 E-Myc transgenic mice were isolated as described previously (Lindemann et al., 2007) and maintained in DMEM supplemented with 10% FCS, 2-mercaptoethanol (50 M), L-asparagin (0.37 mM) and HEPES (pH 7.4, 10 mM). C57BL/6 E-Myc transgenic mice were purchased from the Jackson Laborator.

(13) Reagents and Antibodies:

(14) Rabbit anti-GAPDH was from (Abcam, Cambridge, UK), mouse anti-Erk2 was from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Other antibodies were from Cell Signaling Technology (Beverly, Mass., USA). Mitochondrial complex I inhibitor, phenformin was from Sigma.

(15) Plasmids:

(16) Complementary sense and antisense oligonucleotides were annealed and into BglII/HindIII-cut pSUPER retro.Neo+GFP vector (oligoengine) to produce short hairpin RNA (shRNA) targeting mouse gapdh (shgapdh). shRNA targeting luciferase was used as a control shRNA (shctl).

(17) ATP Analysis:

(18) ATP was measured using the Promega Cell Titer Glo kit. In summary, 20.000 cells were re-suspended in 80 L and distributed in a 96 well plate. Cells were then treated in triplicates with control (PBS), oligomycin A, or sodium iodoacetate both alone or in combination with oligomycin A. Following a 1-hour incubation, 100 uL of cell titer Glo reaction mix were added to each well for a final volume of 200 uL. Plates were then analyzed for luminescence with a Luminoscan. By comparing the different conditions, global ATP and percentages of both glycolytic and mitochondrial ATP were determined.

(19) RNA Extraction and Real-Time Quantitative PCR:

(20) Total RNA was extracted from cells using the RNA extraction kit (Qiagen) according to the manufacturer's instructions. Total RNA (2 g) was added to 20 l reverse transcription-PCR using the Omniscript kit (Qiagen). The relative mRNA expression level of gapdh and ldh-a (mouse) were obtained by real-time quantification PCR (qPCR), using the TaqMan PCR Master Mix (Eurogentec) and TaqMan assay primer set (Applied Biosystems, Foster City, Calif.) on the 7500 Fast and the Step One (Applied Biosystems) according to the manufacturer's instructions (sequences provided upon request). For in vitro experiment, all samples were normalized to 18s.

(21) Western Blot Analysis:

(22) Mouse primary E-Myc cells were washed and lysed in laemmli buffer. Proteins (40 g) were separated on 8% to 12% SDS polyacrilamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were then blotted with antibody corresponding to the indicated proteins. Immunoreactive bands were detected with a horseradish peroxidase (HRP) anti-mouse (Dako) or anti-rabbit (Cell Signaling) by enhanced chemiluminescence (Pierce). When indicated, Western blot quantification was made using ImageJ software.

(23) Proliferation Assay:

(24) E-Myc cells (0.210.sup.6) were seeded in 96 well-plates in the presence or absence of 50 M of Phenformin for 0, 24 and 48 hours. Number of living and dead cells was counted using DAPI staining exclusion by flow cytometry (MACSQuant Analyser, Miltenyi Biotec). Proliferation index were calculated by dividing the number of living cells obtained for each day by the one obtained the day of seeding (day 0).

(25) Cell Death Assay:

(26) E-Myc cells (0.210.sup.6) were seeded in 96 well-plates in the presence or absence of 200 M of phenformin (Sigma) for 24 hours and 48 hours. Cells were then labeled with DAPI (Molecular Probes; 0.5 g/mL) and analyzed immediately by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec).

(27) Animal Studies:

(28) Lymphoma transfer of a isolated E-Myc clone was realized into syngeneic, nontransgenic, 6-week-old C57BL/6 females by tail vein injection of 0.110.sup.6 viable E-Myc lymphoma cells per recipient mouse (in 150 L of sterile PBS). Phenformin treated group received 200 mg/kg/day phenformin in the drinking water for 12 days. Phenformin powder was dissolved every two days in the drinking water, taking into account the mouse body weight and the volume of water consumed each day. Untreated mice (controls) received water (n=10 mice/group). Food was given ad libitum. The first signs of the pathology were determined by inguinal lymph node palpation and analyses of blood sample with Hemavet 950FS (Drew Scientific, INC, France). Progression-free survival was determined by the time between the iv injection of the cell and the appearance of enlarged lympho nodes. Lymphoma-bearing animals were killed by cervical dislocation as soon as they presented signs of illness. Upon sacrifice all lymph node tumors and spleen are immediately collected and weighted. All mice were maintained in specific pathogen-free conditions and experimental procedures were approved by the Institutional Animal Care and Use Committee and by the regional ethics committee (PEA232 from Comit Institutionnel d'Ethique Pour l'Animal de LaboratoireAZUR).

(29) Isolation of Human Tumor Cells from Patient Biopsies:

(30) Tumor biopsy was incubated in RPMI supplemented with 2% of FCS, DNAse (10 g/ml) and collagenase (0.1 mg/ml) for 5 min at 37 C. Tumor cells were then dissociated and incubated in the presence of red blood cell lysis buffer for 1 min. After washing, cells were counted and immediately analyzed for glycolytic vs OxPhos ATP, gapdh mRNA levels (qPCR). Cells were also stained for 30 min with an anti-CD20 antibody (eBioscience, 1/100) to determine the proportion of B cells within the tumor.

(31) Clinical Trial KTMR (Kidrolas-Torisel-Metformine-Rituximab):

(32) Nine R-CHOP refractory Myc+-DLBCL patients with adequate performance status and organ functions were enrolled in a new clinical protocol targeting tumor metabolism. Treatment combined L-asparaginase (Kidrolase, 6000 IU/ml) on days 1, 3, 5, 7, 9, 11 and 13, mTOR inhibitor (Torisel 75 mg/week) on days 1, 7 and 14 and Rituximab (375 mg/m2) on days 1 and 7 of each cycle. In between two cycles of treatment patients received Metformin daily (1000 mg/day). Four patients experienced toxicity and were removed from the study. Three patients completed four cycles of treatment and one patient completed two cycles in complete response. Median duration of response was 6 months (range, 4-6 mo).

(33) Immunohistochemical Staining of GAPDH:

(34) Sections (3 m) of formalin-fixed, paraffin-embedded DLBCL biopsies were treated using standard procedures and were immunostained (using Dako manufacturer instructions) for the expression of GAPDH (Prestige, Sigma, 1/500). GAPDH staining was then scored based on the GAPDH intensity and the percentage of stained cells) to identify GAPDH low and GAPDH high lymphomas.

(35) Statistical Analysis:

(36) Continuous variables and binomial variables, expressed as mean (s.d.), were analyzed with Student's t-test or one-way analysis of variance and compared, respectively, between groups with Fisher's exact test or 2 test. Continuous variables (proliferation index), expressed as mean (s.d.) after log transformation, were analyzed with linear regression and compared between groups with Fisher's exact test. For time-to-event variables, the survival functions were estimated with Kaplan-Meier method and compared with log-rank tests. All statistical analyses were done with R project software (version 2.15.1). A p-value of less than 0.05 was considered to indicate statistical significance (*p<0.05, **p<0.01 and ***p<0.001).

(37) Results

(38) We showed that, in contrast to B lymphomas expressing high levels of GAPDH, B lymphomas expressing low levels of GAPDH or B lymphomas silenced for GAPDH are more sensitive to the mitochondrial complex I inhibitor phenformin. In addition, B lymphoma progression is reduced upon phenformin treatment in E-Myc-gapdh.sup.low-bearing mice.

(39) We first demonstrate that mouse primary E-Myc lymphomas express different levels of gapdh mRNA (FIG. 1A) and GAPDH protein (FIG. 1B), while the other glycolytic enzymes such as HKII, ENO1, LDH-A, PKM2 and PDK1 are expressed to the same extent (FIGS. 1A and 1B).

(40) In contrast to E-Myc-gapdh.sup.high clones which mainly produce glycolytic ATP (70% glycolytic ATP and 30% of mitochondrial ATP), we demonstrated that E-Myc-gapdh.sup.low clones produce 50 to 60% of mitochondrial (OxPhos) ATP (FIG. 2) and that they are more sensitive to mitochondrial complex I inhibitor Phenformin (FIGS. 3A and 3B) and to L-asparaginase (glutamine/asparagine hydrolysis) (FIG. 3C). When E-Myc-gapdh.sup.high cells were specifically silenced for gapdh (by 30%), they were more sensitive to phenformin (FIGS. 4A and 4B) and to L-asparaginase (FIG. 4C) treatment than control (shSCR) E-Myc-gapdh.sup.high cells. In addition, B lymphoma progression is reduced upon phenformin treatment in E-Myc-gapdh.sup.low-bearing mice, as shown by the progression-free survival (FIG. 5A) and by the size of the axillary lymphomas (FIG. 5B) and the spleen (FIG. 5C).

(41) From fresh patient biopsies diagnosed as follicular lymphomas we could confirm that glycolytic tumors express more gapdh mRNA than Oxphos tumors.

(42) Our collaborator Pr. Catherine Thieblemont performed a new clinical protocol targeting tumor mitochondrial metabolism (L-asparaginase+Torisel+Metformine+Rituximab) in which nine R-CHOP-refractory Myc+-DLBCL patients were included. Out of nine patients, three were in complete response (CR), one in partial response, one progressed on therapy and four were removed from the study because of treatment toxicity. Retrospectively, all three DLBCL in CR were expressing low level of GAPDH at diagnosis. One DLBCL expressing low level of GAPDH progressed on therapy.

REFERENCES

(43) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., Boldrick, J. C., Sabet, H., Tran, T., Yu, X., et al. (2000). Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503-511. Monti S, Savage K J, Kutok J L, Feuerhake F, Kurtin P, Mihm M, Wu B, Pasqualucci L, Neuberg D, Aguiar R C, et al. Molecular profiling of diffuse large B-cell lymphoma identifies robust subtypes including one characterized by host inflammatory response. Blood. 2005; 105:1851-1861. Miyoshi H. Structure-activity relationships of some complex I inhibitors. Biochimica et Biophysica Acta (BBA)Bioenergetics 1998 Volume 1364 Issue 2 Pages 236-244. Ramsay E E, Hogg P J, Dilda P J. Mitochondrial metabolism inhibitors for cancer therapy. Pharm Res. 2011 November; 28(11):2731-44. Wise D R, Thompson C B. Glutamine Addiction: A New Therapeutic Target in Cancer Trends Biochem Sci. 2010 August; 35(8):427-33.