ALBERT

Description: Introduction ASCT is a cornerstone in lymphoma therapeutics, especially in the relapse setting. BEAM (carmustine, etoposide, cytarabine, and melphalan) is the most widely used conditioning regimen with a low rate of transplant mortality. Bendamustine has occasionally been used in France as a substitute for carmustine during a period of shortage ("BendaEAM"). SOS is a potentially life-threatening complication, rarely observed in ASCT. In an EBMT study of 1010 ASCT, SOS occurred in less than 1% after BEAM in patients who had a normal level of AST before conditioning1. After having observed 5 cases of SOS in our institution during a 3 year period and numerous cases in other centers, we did perform a retrospective study in order to try to understand the apparent increase in incidence of this complication. Methods After a national alert spread through our cooperative group (LYSA) and the French health agency (ANSM), we collected history of lymphoma, hepatic disease, chemotherapy, SOS risks factor, conditioning of ASCT, diagnostic criteria for SOS (based on the EBMT classification2 or liver biopsy), treatment and outcomes. We did determine the incidence in 2 centers (Henri Mondor University Hospital and Curie Institute) where the total number of ASCT was available. Results We observed 6 cases of SOS after ASCT in Henri Mondor University Hospital during the last 10 years (incidence 4.1% on 145 ASCT for lymphoma) among which the 5 most recent cases had oxaliplatin exposure before ASCT. Oxaliplatin exposition tended to be associated with SOS (p=0.09). In Curie Institute, a SOS incidence of 3/46 (6.5%) was reported during a 6 year-period, and all three patients had received oxaliplatin before ASCT. A national query permitted to find 22 cases of SOS in 10 centers during the last 6 years. Twenty had a history of oxaliplatine exposure before ASCT. Principal characteristics are presented in table 1. No patient had a history of cirrhosis or significant hepatic pathology. Two patients had liver infiltration due to the lymphoma before treatment by oxaliplatin. The majority of patients didn't present with any of the known risk factors for SOS. One patient had liver anomalies (AST 〉 2 ULN and cholestasis) after 4 cycles of an oxaliplatin-containing regimen for relapsing diffuse large B-cell lymphoma. Biopsy of liver showed early sinusoidal lesions. Despite these results, he proceeded to ASCT with BEAM conditioning and experimented a moderate form of SOS. Five patients received fluconazole and aprepitant during conditioning. Discussion During the last decade, cisplatin was often substituted by oxaliplatin inside the DHAP protocol in order to avoid renal impairment. Pre-operative oxaliplatin in hepatic metastases of colorectal cancer leads to asymptomatic damage of sinusoidal endothelial cells (SEC) in half of the patients3. Our principal hypothesis is that oxaliplatin provokes a frailty of liver and conditioning of ASCT and co-factors (peg-filgrastim and cytochromes inhibitors- fluconazole and aprepitant) lead to SOS. SEC had a receptor for G-CSF and activation of this receptor lead to inflammatory state4. Peg-filgrastim administered at a fixed dose in the early time of ASCT (Day+1 or +3) with low concurrence increases the inflammatory state of SEC. Fluconazole and aprepitant are inhibitors of cytochromes 3A4 which metabolize etoposide. Each inhibitor could increase exposition to etoposide. Five patients received both inhibitors during conditioning which might have increased liver toxicity which led to SOS. Conclusion We want to warn about the probable association of oxaliplatin before ASCT and SOS. In some French centers, this led to switch oxaliplatin by carboplatin for patients eligible to ASCT. BibliographyCarreras E et al. Incidence and outcome of hepatic VOD after blood or marrow transplantation: a prospective cohort study of the EBMT. Blood. 1998;92(10):3599-3604.Mohty M et al. Revised diagnosis and severity criteria for SOS/VOD in adult patients: a new classification from the EBMT. Bone Marrow Transplant. 2016;51(7):906-912.Rubbia-Brandt L et al. Severe SOS associated with oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer. Ann Oncol. 2004;15(3):460-466.Fusté B et al. GSF factor increases expression of adhesion receptors on endothelial cells through activation of p38 MAPK. Haematologica, 2004;8. Table 1. Table 1. Disclosures Tilly: Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria. Haioun:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Sciences: Consultancy, Honoraria.

Description: Virtually all recurrent molecular alterations in acute myeloid leukemia (AML) functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as patients, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, as demonstrated by western blotting, immunofluorescence, flow cytometry and electron microscopy. Blocking autophagy via shRNA-mediated knockdown of ATG5 and ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation in AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1 both directly (through raptor phosphorylation) and indirectly (through TSC2 phosphorylation), sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. This protective effect was mediated by mTORC1-dependent modulation of the ATF4/CHOP stress response pathway. The ultimate functional consequence was that, rather than diminishing GSK621-induced cytotoxicity, persistent mTORC1 activity was in fact synthetically lethal with AMPK activity in AML cells. This synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. It could also be enhanced in AML cells by mTORC1 overactivation induced by CRISPR-mediated deletion of TSC2. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. This finding may have important implications for AMPK and mTORC1 signaling pathways in cancer biology more broadly. Context-dependent permissiveness towards mTORC1 activation may amplify the response to cytotoxic stress, such as that resulting from AMPK activation by GSK621. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials. Disclosures Brusq: GSK: Employment. Nicodeme:GSK: Employment.

Description: Abnormal activation of kinases may promote leukemogenesis by conferring cell proliferation and survival advantage in acute myelogenous leukemia (AML). New targeted therapies are currently developed to disrupt such kinases activation to improve the long term prognosis of AML patients. However, the prognosis impact on AML patients has only been evaluated for a few kinases, including FLT3 and ERK/MAPK. Recent reports suggest that PI3K activation may confer poor survival to AML patients. Pre-treatment primary bone marrow blast cells samples from 92 patients registered for the LAM2001 trial of the French GOELAMS group were analyzed and PI3 kinase constitutive activation was correlated with outcome. As previously reported (V Bardet, Haematologica, 2006, 91, 757–764), the status of AKT phosphorylation on Ser 473 was determined using either Western Blot analysis (in samples 〉90% blasts) or flow cytometry in the CD45Low positive population of blast cells. Phosphorylation of AKT on Ser473 was determined after a 4 hour period of deprivation in cytokine and serum-free medium. In such conditions, we can clearly define to groups of patients, one with constitutive activation of PI3K (PI3K+) and the other without detectable phosphorylation of AKT (PI3K−). At inclusion, 42 patients were males and 50 were females. Median age at diagnosis was 46 (17,3–64,5) years. Leukocytosis was 12000/μL (range 400–251800). Patients were classified in low-risk, standard-risk and high-risk cytogenetic subgroups in respectively 16%, 61% and 23%. 16/61 (25%) had FLT3-ITD mutation, 9/60 (15%) had Ras (N-Ras or K-Ras) mutations and 15/59 (25%) had NPM mutations. 46 of 92 (50%) patients had constitutive PI3K activation (PI3K+ group). No difference was observed between the PI3K+ and PI3K− groups for all previously descripted parameters, except concerning FLT3 mutational status: 5/32 (16%) PI3K+ patients had FLT3-ITD versus 11/29 (38%) in the PI3K− (p = 0,048). All patients received one course of induction chemotherapy (3+7 regimen), and 31 (34%) a second induction course at day 15. Complete remission was obtained in 74 patients (80%) with no difference between the two groups (p = 0,60). Among responders, 30 (41%) had autologous stem cell transplantation, 24 (32%) had allogenic bone marrow transplantation and 16 (22%) had consolidation chemotherapy. With a median follow-up of 26 months (range 3 days–37 months), 48 patients died. Overall survival was 56% in the PI3K+ group and 33% in the PI3K− group (p=0.007). Among responders, 30 patients had relapse. Relapse free survival was 72% in the PI3K+ group and 41% in the PI3K− group (p=0.001). Thus constitutive PI3K activity detected in leukemic cells at diagnosis confer a better prognosis in AML. We observed significantly more relapses in PI3K− patients. These results suggest that PI3K activity in AML may confer a particular sensitivity to chemotherapy, the demonstration of which remain to be made. Furthermore, inhibition of PI3K signaling pathway may not be as beneficial as expected, and a better understanding of downstream effectors is clearly needed before targeting PI3K in AML.

Description: Key Points Glutamine removal and knockdown of the glutamine transporter SLC1A5 have antileukemic activity in AML. The glutaminase activity of l-asparaginase inhibits mTORC1 and protein synthesis and induces a strong autophagy in AML.

Description: Nucleoside analogs (NA) are used alone or in combination in low grade B-cell lymphoproliferative disorders for 20 years. The incidence of disease transformation (Richter syndrome: RS) and development of MDS/AML among patients (pts) with indolent B-cell malignancies receiving NA are still controversial. We have reviewed the crude incidence of RS and MDS/AML in 165 WM patients treated with 3 F-based regimens. Results: Retrospective Study 1 (Leblond J Clin Oncol 1998): F (25mg/m2 iv D1–5) in 71 WM pts in relapse/refractory treated with alkylator, median time from diagnosis (dg) to tt: 5.9yrs, median prior tt: 2 (1–4). Randomized prospective study 2 (Leblond Blood 2001) in 92pts in first relapse after alkylator: Arm 1: F (25mg/m2 ivD1–D5) (45pts) vs Arm 2: CAP (doxorubicine 25mg/m2 IV D1, cyclophosphamide 750mg/m2 IV D1, Prednisone 40mg/m2 D1–5) (45pts), median time from dg to tt: 3.9yrs. Retrospective study N°3 (Tamburini Leukemia 2005) in first-line WM (14pts) or relapse WM (35pts): F 30mg/m2 D1–3 by oral route + Cyclophosphamide (C) 300mg/m2 D1–3 by oral route, median time from dg to tt: 2.1 yrs. The incidences of MDS/AML and RS are shown in Table 1 Discussion: the crude incidence of RS varied from 6,6% to 8% and was not statistically different in the randomized prospective study N°2 between the two groups. However, the incidence reported in pts treated with only alkylating-based regimens was lower: 3/167(1.8%) (Facon J Clin Oncol1993) and 3/217 (1.4%) (Garcia Sanz Br J Hematol 2001) and could be explained by a F use as salvage treatment in the CAP arm. The impact of fludarabine on RS needs to be confirmed in larger prospective studies. The crude incidence of MDS/AML varied from 1.4 to 8.9% in pts treated with fludarabine alone and was 6% in pts treated with F+C. The incidence reported in pts treated with alkylating based regimens varied from 1% (2/167, Facon 1993, 3/217 Garcia-Sanz 2001) to 6% (3/46, Kyle Br J Hematol 2000,) and was not different. In other low grade B-cell malignancies, a higher incidence of MDS/AML has been reported in pts treated with analog-alkylator combination compared to F or alkylator alone (Morrison J Clin Oncol 2002, Bowcock Br J Hematol 2006, Tam Hematologica 2006). In our study, the rate was the same in the two populations. The impact of late effects, particularly second malignancies, need to be better evaluated in prospective studies, especially in young patients and clinicians should discuss the risk when recommending such therapy to pts. MDS/AML and RS incidences in the three studies Study N°, pts Median survival time from study Median follow-up time from study MDS/AML (N,%) RS (N,%) N°1 71 pts 23 mo 34 mo 1/71 (1.4%) 5/71 (7%) N°2- 92 pts 41mo( arm1), 45 mo (arm2) 34mo 4/45 (8.9%, arm1), 2/45( 4.5%, arm2) 3/45 (6.6% arm1), 2/45 (4.5%, arm2) N°3-49pts 64% at 60 mo 42 mo 3/49 (6%) 4/49 (8%)

Description: The phosphatidylinositol 3-kinase (PI3K)/Akt and mTORC1 pathways are frequently activated, representing potential therapeutic targets in acute myeloid leukemia (AML). In 19 AML samples with constitutive PI3K/Akt activation, the rapamycin derivative inhibitor everolimus (RAD001) increased Akt phosphorylation. This mTOR C1-mediated Akt up-regulation was explained by an insulin-like growth factor-1 (IGF-1)/IGF-1 receptor autocrine loop: (1) blast cells expressed functional IGF-1 receptors, and IGF-1-induced Akt activation was increased by RAD001, (2) a neutralizing anti-IGF-1R α-IR3 monoclonal antibody reversed the RAD001-induced Akt phosphorylation, and (3) autocrine production of IGF-1 was detected in purified blast cells by quantitative reverse transcription-polymerase chain reaction and immunofluorescence. This RAD001-induced PI3K/Akt up-regulation was due to an up-regulated expression of the IRS2 adaptor. Finally, we observed that concomitant inhibition of mTORC1 and PI3K/Akt by RAD001 and IC87114 induced additive antiproliferative effects. Our results suggest that dual inhibition of the mTORC1 complex and the IGF-1/IGF-1R/PI3K/Akt pathway in AML may enhance the efficacy of mTOR inhibitors in treatment of this disease.

Description: Acute myeloid leukemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) is a poor prognosis hematologic malignancy accounting for 30% of AML cases. Constitutive FLT3-ITD activation drives STAT5 signaling resulting in enhanced PIM kinases expression. PIM serine/threonine kinases (including PIM1,-2,-3) are involved in cell cycle and apoptosis regulation and thus represent emerging therapeutic targets. We previously reported an increased PIM2 protein expression in primary AML cells compared to normal CD34+ immature hematopoietic cells. Here, we aimed to study PIM kinases as potential therapeutic target in FLT3-ITD AML. In two distinct FLT3-ITD+ human AML cell lines (MV4-11 and MOLM-14) doxycycline (Dox)-induced shRNA-mediated PIM2 knockdown enhanced apoptosis, attested by an increase in early apoptotic (annexin V positive, DAPI negative) and late apoptotic (both annexin V, DAPI positive) cells. Cell death upon PIM2 knockdown was confirmed by an inhibition of colony formation in methylcellulose. Mechanisms of apoptosis induction involved release of second mitochondria-derived activator of caspases (SMAC) as well as increased p53 and Bax expression and Bax nuclear translocation, leading to loss of mitochondrial membrane potential. To gain further mechanistic insights, we performed global gene expression profiling in the MOLM-14 cell line lentivirally transduced with Dox-inducible PIM2 shRNA. Consistent with our functional analysis, cell-cycle regulatory genes (including c-MYC, CHK1 or PLK1) and pro-survival genes (including RSK2 or BCL2) were down-regulated, while pro-apoptotic genes (most notably TP53 and BAX) were up-regulated. Here we focused on RSK2, a member of the mitogen-activated protein kinase (MAPK) signaling pathway that has not been previously reported to be a target of PIM2. In the MOLM-14 cell line, PIM2 knockdown reduced RSK2 mRNA and protein levels. RSK2 knockdown using a Dox-inducible RSK2 shRNA induced apoptosis similarly to that observed following PIM2 knockdown in this cell line. In parallel with PIM2 knockdown, RSK2 down-regulation enhanced p53 expression and activity, as measured by increased expression of its transcriptional target p21, as well as Bax expression. In summary, our results suggest that RSK2 is a PIM2 transcriptional target that contributes to PIM-2-dependent cell survival mediated by a novel RSK2-p53-Bax signaling pathway. RSK2 therefore warrants further study as a potential pharmacologic target in AML (particularly in cases resistant to PIM inhibition) as well as other PIM-addicted malignancies. Disclosures No relevant conflicts of interest to declare.

Description: To expedite the translation of biologic discoveries into novel therapeutics, there is a pressing need for panels of in vivo models that capture the molecular complexity of human disease. While traditional cell lines and genetically engineered mouse models are useful tools, they are insufficient to assess the broad diversity of human tumors within a context that recapitulates in situ biology. Patient-derived xenografts (PDXs), generated by transplanting primary human tumor cells into immune-deficient NOD.Cg-Prkdcscid/Il2rgtm1Wjl/SzJ (NSG) mice, surmount some of the limitations of these traditional platforms and have been increasingly utilized as tools for preclinical investigation. However, the infrastructure required to generate, bank, and characterize PDX models limits their availability to only a few investigators. To address this issue, we established a repository of PDX models of leukemia and lymphoma, which we have named the Public Repository of Xenografts (PRoXe). At the time of this writing, PRoXe contains 213 independent lines that have been passaged through mice once (P0), 123 of which have been repassaged in a second generation (P1) or further repassaged. The repository encompasses AML, B- and T-ALL, and B- and T-cell non-Hodgkin lymphoma (NHL) across a range of cytogenetic- and molecularly-defined subtypes (Table 1). PRoXe is extensively annotated with patient-level information, including demographics, phase of treatment, prior therapies, tumor immunophenotye, cytogenetics, and molecular diagnostics. PDX lines available for distribution are characterized by immunophenotyping, whole transcriptome sequencing (RNAseq), and targeted exon sequencing of ~300 genes. To confirm fidelity of engrafted tumors to their corresponding clinical samples, lymphomas were morphologically assessed in P0 mice by H&E and, when pathologic adjudication was required, by immunohistochemistry. Xenografted leukemias were compared to their original tumors immunophenotypically. Unsupervised hierarchical clustering was performed on 132 of these lines based on transcriptome sequencing data and demonstrated 94% concordance between classification of the PDX lines by RNA expression and by the annotated clinical-pathologic diagnoses. Discordant cases highlighted unusual variants, such as B-ALL with aberrant expression of myeloid markers and a follicular lymphoma that underwent blastic transformation in the mouse. Multiple lines have been luciferized and confirmed to home to bone marrow, spleen, and liver. Existing lines from PRoXe have already been shared with more than ten academic laboratories and multiple industrial partners. All of the data referenced here are freely available through a customized web-based search application at http://proxe.org, and lines can be requested for in vitro or in vivo experiments. We are actively expanding the size of PRoXe to allow for large pre-clinical studies that are powered to detect differences across genetically defined subsets. Thus, we are happy to host additional lines from outside investigators on PRoXe and thereby expand the availability of these valuable reagents. Finally, we have made the source code for PRoXe (in R Shiny) open-access, so that other investigators can establish their own portals. Table 1. WHO diagnostic entities encompassed within PRoXe at P1 or later, or P0 or later for B-ALLs. WHO Classification - number of lines per diagnostic entity AML, Other Myeloid, and Ambiguous Lineage [n=32] ALL [n=107] AML - recurrent gene mutations 6 B-ALL - NOS 44 AML - MDS-related changes 5 B-ALL - MLL-rearranged 11 AML - NOS 4 B-ALL - BCR-ABL 10 AML - MLLT3-MLL 2 B-ALL - hyperdiploidy 9 Acute myelomonocytic leukemia 1 B-ALL - TEL-AML1 8 Acute monocytic leukemia 1 B-ALL - E2A-PBX1 3 AML unable to classify 2 B-ALL unable to classify 1 Blastic plasmacytoid dendritic cell neoplasm 8 T-ALL 21 Mixed phenotype, MLL rearranged 1 B/myeloid acute leukemia 1 Myelodysplastic syndrome 1 Mature B cell neoplasms[n=11] Mature T and NK cell neoplasms [n=4] DBLCL - NOS 4 Angioimmunoblastic T-cell lymphoma 1 Mantle cell lymphoma 3 Adult T-cell leukemia/lymphoma 1 Extranodal marginal zone lymphoma 1 Extranodal NK/T-cell lymphoma 1 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL 3 SŽzary syndrome 1 Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Etchin:Karyopharm: Research Funding. Lane:Stemline Therapeutics, Inc.: Research Funding. Stone:Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Merck: Consultancy; Karyopharm: Consultancy; Roche/Genetech: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy.