Pharmacological Induction of NOXA Sensitizes High-Risk B Cell Acute Lymphoblastic Leukemia Cells to Venetoclax

Background: Venetoclax (VEN), a specific BCL2 inhibitor, exerts anti-leukemic effects in various high-risk (HR) B-ALL subtypes, such as ALL with mixed lineage leukemia (MLL) gene rearrangements (MLLr ALL) (PMID: 26711339), Philadelphia chromosome-positive (Ph+) (PMID 30546081) or hypodiploid B-ALL (PMID 30862722). Nevertheless, despite high rationale for targeting BCL2 in these subtypes of B-ALL, VEN monotherapy is not effective enough to completely eliminate leukemic cells. For this reason identification of other drugs that could sensitize leukemic cells to VEN may become beneficial treatment strategy in HR ALL. Previously, we showed that the enzymes of the thioredoxin system are upregulated in primary B-ALL cells and that auranofin (AUR), a thioredoxin reductase inhibitor, effectively kills leukemic cells in vitro and in vivo. Importantly, elements of the thioredoxin system are not only balancing redox homeostasis within the cells, but may also interact with other pathways, including anti-apoptotic signaling. Considering above, we hypothesized that AUR may potentiate VEN efficacy in HR B-ALL. Methods: To evaluate cytostatic/cytotoxic effects of VEN+AUR combination by MTT assay and propidium iodide (PI)-staining we used HR B-ALL cell lines, including SEM (MLLr ALL), BV-173 (Ph+ ALL) and NALM-16 (hypodiploid ALL). Patient derived xenograft cells (PDX) were generated through long-term propagation of primary B-ALL samples in immune-deficient NSG mice. Ex vivo drug testing in co-culture system was performed using primary bone marrow-derived mesenchymal stem cells (BM-MSC) and murine stromal OP9 cell line. NOXA genomic knockout (KO) in SEM cells was established by CRISPR/Cas9 system. Chromatin accessibility within PMAIP1 gene (encodes for NOXA) was detected using ATAC-seq. Results: We observed that AUR sensitizes HR B-ALL cell lines to VEN, as determined by MTT and PI-staining. Further, we mimicked the bone marrow support of stromal cells towards B-ALL and evaluated its impact on the response to VEN+AUR. For this reason we employed an ex vivo co-culture system of B-ALL PDX cells with primary BM-MSC or an OP9 cell line. In all tested PDX samples representing diagnostic/relapsed MLLr ALL (n=8), Ph+ ALL (n=2) and Ph-like ALL (n=2) we observed synergistic effect of this combination (Fig. 1A). Next, we determined the efficacy of VEN+AUR combination in vivo using a PDX model of MLLr B-ALL. We observed that administration of VEN+AUR diminished the progression of leukemia during a 3 week-long treatment more effectively than any single drug alone, which reflected in longer survival of NSG mice (Fig. 1B). Subsequently, we aimed to uncover the mechanism responsible for the synergistic action of VEN+AUR. In cells treated with both drugs we observed enhanced caspase activation and changes in the levels of BCL2 family proteins involved in apoptotic signaling. In particular, we found that AUR strongly upregulates a pro-apoptotic NOXA protein, both in HR B-ALL cell lines and in MLLr ALL PDX samples (Fig. 1C). To evaluate whether NOXA induction is functionally relevant for the cell death mediated by VEN+AUR, we generated SEM cells with a NOXA genomic KO. Lack of NOXA significantly abolished VEN-single agent as well as VEN+AUR combination cytotoxicity, demonstrating its dependence on NOXA expression (Fig. 1D). We then showed that NOXA is regulated at the transcriptional level, as co-treatment with AUR and the transcription inhibitor, actinomycin D, abolished AUR-mediated NOXA induction at mRNA and protein levels in SEM cells. Additionally, to test whether AUR-treatment itself provokes changes in chromatin accessibility within the NOXA encoding gene (PMAIP1) we performed ATAC-seq. We observed a clear increase in accessibility at PMAIP1 in response to AUR, which correlated with transcriptional induction of NOXA. Moreover, ChIP-qPCR revealed that increased ATAC peaks within PMAIP1 were associated with an increase in H3 lysine 27 acetylation (H3K27ac) - an epigenetic mark associated with open chromatin conformation. Conclusions: Our results demonstrate that FDA-approved drug, AUR, is a promising candidate to be used in combination with VEN for the therapy of HR B-ALL subtypes. Importantly, NOXA induction by AUR plays a central role in the VEN+AUR synergistic cytotoxicity. More studies elucidating the mechanism of NOXA upregulation by AUR are underway. Disclosures Milne: OxStem Oncology (OSO), a subsidiary company of OxStem Ltd.: Other: Founding shareholder .

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BCL-2 Inhibition As a Synthetic Lethal Approach To Target Isocitrate Dehydrogenase Mutations In Acute Myeloid Leukemia Stem Cells

Blood ◽

10.1182/blood.v122.21.885.885 ◽

2013 ◽

Vol 122 (21) ◽

pp. 885-885 ◽

Cited By ~ 2

Author(s):

Steven M. Chan ◽

Bruno C Medeiros ◽

Ravi Majeti

Keyword(s):

Cell Lines ◽

Ex Vivo ◽

Leukemic Cells ◽

Synthetic Lethal ◽

Nsg Mice ◽

Idh Mutations ◽

Selective Elimination ◽

Shrna Library ◽

Mutant Idh1

Abstract Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in about 15% of acute myeloid leukemia (AML) patients. Studies of paired samples at diagnosis and relapse have demonstrated that IDH mutations, in contrast to FLT3 or RAS mutations, are stable during disease evolution. This finding indicates that a small population of cells harboring IDH mutations can persist in remission and eventually contribute to relapse. Therapeutic strategies that eradicate this IDH-mutated population have the potential to result in long-term remission. Small molecule inhibitors specific for the mutant IDH enzymes have recently been developed (Wang et al Science 2013), but it is not known if they are effective in targeting primary AML cells including the leukemic stem cell (LSC) compartment in vivo. We sought an alternative approach to target IDH-mutated cells based on the concept of non-oncogene addiction which refers to the increased dependence on a subset of non-mutated genes for survival in response to activation of a specific oncogene. This dependency can be exploited therapeutically by inhibiting the activity of these non-oncogenes resulting in selective elimination of malignant cells, a phenomenon known as synthetic lethality (SL). In an effort to identity SL targets against IDH mutations, we performed a pooled lentiviral RNA interference (RNAi) screen to search for genes that, when inhibited, led to the selective elimination of mutant IDH1 expressing cells. Our lentiviral short hairpin RNA (shRNA) library consisted of 27,500 unique shRNAs targeting 5,043 human genes of relevance to cancer biology. Each shRNA was tagged with a unique barcode sequence which permitted downstream identification by sequencing. The lentiviral shRNA library was transduced into a human AML cell line engineered to express mutant IDH1 (R132H) under the control of a doxycycline-inducible promoter. Following transduction, the cells were cultured in doxycycline to deplete shRNAs that were synthetic lethal to mutant IDH1. The relative abundance of each shRNA was subsequently determined by high-throughput sequencing of the barcode. Using a stringent algorithm designed to minimize false positive hits, the prosurvival gene BCL-2 was identified as one of the top SL hits. We confirmed that RNAi-mediated knockdown of BCL-2 expression was selectively lethal to mutant IDH expressing AML cell lines and further demonstrated that exposure to a cell-permeable form of (R)-2-hydroxyglutarate, the oncometabolite produced by mutant IDH, was sufficient to induce BCL-2 dependence. Similarly, pharmacologic inhibition of BCL-2 with ABT-199, a novel orally bioavailable and highly specific inhibitor of BCL-2 (Souers et al Nature Medicine 2013), was significantly more toxic to mutant IDH expressing AML cell lines than isogenic cell lines with wildtype (WT) IDH. We next investigated the impact of IDH mutation status on ABT-199 sensitivity of primary AML cells and found that FACS-purified blasts with IDH mutations were 10-fold more sensitive to ABT-199 than blasts with WT IDH in ex vivo culture conditions. Normal cord blood hematopoietic stem and progenitor cells were highly resistant to ABT-199 treatment ex vivo suggestive of a wide therapeutic index. To demonstrate in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with primary human IDH-mutated leukemic cells with either ABT-199 at a dose of 100 mg/kg/day or vehicle control for 7 consecutive days by oral administration. Bone marrow engraftment analysis before and after treatment showed a 10 to 20-fold reduction in leukemic burden in ABT-199 treated mice, whereas no difference was seen in vehicle-treated mice. Importantly, bone marrow cells collected from ABT-199 treated mice failed to engraft in secondary transplant recipients indicative of a loss of LSC activity. In separate experiments, lentiviral transduction of BCL-2 shRNA vectors into IDH-mutated primary AML cells to knockdown BCL-2 expression impaired their engraftment in NSG mice, further validating the detrimental effect of BCL-2 inhibition on IDH-mutated LSCs. In summary, our results indicate that IDH mutations increase BCL-2 dependence in leukemic cells including LSCs and identify a subgroup of patients that is likely to respond to pharmacologic BCL-2 inhibition. Our data provide the preclinical rationale for investigating the use ABT-199 in this patient subgroup in clinical trials. Disclosures: No relevant conflicts of interest to declare.

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CD46 Is Amplified in High-Risk Myeloma with Gain of Chromosome 1q and Selectively Targeted By a Novel Anti-CD46 Antibody-Drug Conjugate

Blood ◽

10.1182/blood.v128.22.384.384 ◽

2016 ◽

Vol 128 (22) ◽

pp. 384-384

Author(s):

Daniel W. Sherbenou ◽

Blake T. Aftab ◽

Yang Su ◽

Christopher R. Behrens ◽

Arun P. Wiita ◽

...

Keyword(s):

Single Dose ◽

High Risk ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Ex Vivo ◽

Chromosome 1Q ◽

Nsg Mice ◽

Antibody Drug

Abstract Background: Multiple myeloma (MM) is incurable by standard approaches, with relapse and development of treatment resistance inevitable in all patients. We previously identified a panel of novel macropinocytosing human monoclonal antibodies against CD46 by phage antibody library display and optimized a lead antibody for targeted drug delivery. Antibody-drug conjugates (ADCs) have recently seen proof-of-concept clinical success in Hodgkin lymphoma and breast cancer, but none is yet FDA-approved for MM. The CD46 gene is located on the long arm of chromosome 1 (1q32.2), 50Mbp from a FISH probe clinically used to identify high-risk MM and which may provide a surrogate biomarker for CD46 as a therapeutic target. Methods:We covalently conjugated the monomethyl auristatin F (MMAF) toxin to our anti-CD46 antibody via a lysosomal protease sensitive valine-citrulline linker (hereafter referred to as CD46-ADC). High Performance Liquid Chromatography analysis with hydrophobic interaction chromatography of the final conjugate showed an average drug per antibody of 3.3. CD46-ADC was evaluated for cytotoxicity in vitro in MM cell lines, in vivo with cell line xenografts in NSG mice, and ex vivo in MM patient bone marrow (BM)aspirate samples. To assess in vivo toxicity, CD46-ADC treatment was administered to transgenic mice that express the human CD46 gene under its native promoter. Results: CD46 was highly expressed on the cell surface of all 18 MM cell lines tested, and was upregulated on MM1.S cells co-cultured with the BM stromal cell line HS5. In BM aspirate samples, CD46 was highly expressed on MM cells in 100% (n=25) patients evaluated. By quantitative flow cytometry in 10 patients, the CD46 cell surface antigen density was significantly higher in patient MM cells with 1q21 gain (1q+) than those with normal 1q21 copy number (p=0.032) (Fig 1A). In patients with amp1q21 the mean CD46 antigen density on MM cells was 313,190 (SEM 68,849), compared to patients with normal 1q21 where it was 121,316 (SEM 28,352) (Fig 1A). In contrast, CD46 antigen density on normal donor (n=3) BM hematopoietic cell populations was low (antigen density range 8,443 - 23,772). Of note, higher CD46 antigen density was present on monocytes (mean 58,320, SEM 6,874) and granulocytes (mean 54,439, SEM 10,688) relative to the other populations (Fig 1B). CD46-ADC potently inhibited proliferation in all 14 MM cell lines tested (EC50 range of 150 pM - 5 nM) (Fig 1C). On BM stromal cells, CD46-ADC had EC50 >100 nM for patient-derived BM61 (generated via culture of CD138-negative BM) cells and no effect on HS5 cells in concentrations tested up to 150 nM. CD46-ADC eliminated MM growth in two orthometastatic xenograft models. In one model, MM1.S cell line xenografts expressing firefly luciferase grown in NSG mice were treated once every 3-4 days at either 4 mg/kg or 0.8 mg/kg for 4 injections, or with a single dose of 4 mg/kg (Fig 2A). Control groups were treated with vehicle, nonbinding ADC or naked antibody (CD46-mAb). CD46-ADC 4 mg/kg (4 dose) eliminated bioluminescent activity throughout the duration of the study (Fig 2B), and all mice survived to study discontinuation (Fig 2C). The single dose and low dose groups showed elimination of bioluminescence, but all mice relapsed (Fig 2B-C). In patient BM aspirate samples, CD46-ADC induces apoptosis and cell death in primary MM cells ex vivo (EC50 <10 nM), but did not affect the viability of non-tumor mononuclear cells (MNCs). For in vivo toxicity study, human CD46 transgenic mice were treated with a single IV bolus injection of 6 mg/kg CD46-ADC and showed no body weight loss or overt side effects for 14 days. At study discontinuation (day 14), histologic analysis of major organs showed no notable tissue damage. Conclusion: We have identified a novel functional antigen, CD46, for ADC targeting of MM, with unique potential for high-risk and relapsed/refractory disease that has genomic amplification at the CD46 gene locus and are in dire need of therapy. The novel CD46-ADC is highly potent and selective in eliminating MM cells (cell lines and primary tumor cells) in preclinical models. CD46 genomic gain on chromosome 1q correlates with antigen amplification, andindentifies a potential biomarker based on a clinical FISH test that can be used for patient stratification. Thus, our study could lead directly to the application of a novel ADC therapeutic for treating MM. Disclosures Aftab: Onyx Pharmaceuticals, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Omniox, Inc.: Research Funding; CytomX: Research Funding; Cleave Biosciences, Inc.: Research Funding. Wiita:Onyx Pharmaceuticals: Research Funding; Omniox, LLC: Research Funding; Cleave Biosciences: Research Funding; Quadriga Biosciences: Research Funding. Wolf:Celgene: Honoraria; Telomere Diagnostics: Consultancy; Takeda: Honoraria; Amgen: Honoraria; Pharmacyclics: Honoraria. Martin:Sanofi: Research Funding; Amgen: Research Funding.

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Next Generation XPO1 Inhibitor KPT-8602 for the Treatment of Drug-Resistant Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.1818.1818 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1818-1818 ◽

Cited By ~ 2

Author(s):

Joel G Turner ◽

Jana L Dawson ◽

Christopher L Cubitt ◽

Erkan Baluglo ◽

Steven Grant ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Ex Vivo ◽

Single Agent ◽

Resistant Cell ◽

Drug Resistant ◽

Newly Diagnosed ◽

Induced Apoptosis ◽

Drug Resistant Cell

Abstract Purpose Human multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects. Experimental Procedures To test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry. Results Viability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples. Conclusions We found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials. Disclosures Shacham: Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.

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The Pro-Tumorigenic Vascular Niche Sustains the T-Cell Acute Lymphoblastic Leukemia Phenotype and Fosters Resistance to Therapy

Blood ◽

10.1182/blood.v128.22.279.279 ◽

2016 ◽

Vol 128 (22) ◽

pp. 279-279

Author(s):

Filomena Di Giacomo ◽

Xujun Wang ◽

Danilo Fiore ◽

Lorena Consolino ◽

Jude Phillip ◽

...

Keyword(s):

High Risk ◽

Cell Lines ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Serum Cytokine ◽

Nsg Mice ◽

Cell Acute Lymphoblastic Leukemia ◽

Free Media

Abstract Introduction. T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy associated with a high risk of treatment failure. Efforts to improve outcomes have focused on underlying genetic defects. However, new evidence suggests that the microenvironment can foster drug resistance/relapses. Identification of factors that contribute to microenvironment-mediated chemo-refractoriness remains an important challenge. Here, we sought to construct an in vitro platform to dissect tumor-host interactions and to optimize drug treatments using Patient-Derived Tumor Xenograft models (PDTX) of high risk adult T-ALL and engineered human endothelial cells. Methods. T-ALL PDTX were established and serially passaged in NSG mice. Engraftment was monitored by flow cytometry of peripheral blood and/or MRI. Mice were sacrificed and leukemic cells were harvested from the spleen/bone marrow. To determine the ex vivo growing conditions, we first cultured a panel of 8 "bona fide" T-ALL cell lines and 11 PDTX cells alone in complete RPMI 20% FCS supplemented with IL2, IL12, IL15 and IL7; or co-cultured with human E4-ORF1 endothelial cells (ECs) without ILs in complete RPMI 20% FCS or serum/cytokine-free media. CDK4/6, MEK, PI3K and JAK inhibitors were used at 0.1 and 1 µM alone and in combination. Cell titer glo, cell titer blue, Annexin-V and S-cell cycle analysis were used as readouts. Total RNA from cells before and after co-culture was extracted for paired-end RNA sequencing on an Illumina HiSeq2500. Results. To study the supporting role of ECs, we first co-cultured ECs with T-ALL cell lines in vitro (serum/cytokine free co-culture) and showed that ECs could reproducibly sustain the viability of 3/8 cell lines (Loucy, KOPTK1, P12 Ichikawa) serum/cytokine-free media. A partial rescue was seen with 3 additional lines (HPB-ALL, CCRF-CEM, CUTLL1), while 2 (KE37, DND41) underwent massive cell death. We next tested whether either ILs or CXCL12 could provide anti-apoptotic signals and demonstrated that KOPTK1 and Loucy were only partially rescued by IL15 or CXCL12. Conversely, IL7, although capable of inducing a robust upregulation of pSTAT5, had no effect (CCRF-CEM and CUTLL1). We then characterized 11 PDTX from 15 high-risk adult T-ALL patients. All PDTX were serially propagated and caused T-ALL in subsequent NSG mice (massive spleen and bone marrow infiltration with extensive paravertebral mass associated with paralysis and multi-organ involvement). Genomic analysis (RNA-seq) demonstrated a high concordance between primary (pre-implant) and PDTX samples. All of them were extensively studied ex vivo, demonstratingthat T-ALL PDTX cells could only survive in ILs supplemented media, even better if enriched of growth factors and supplements for the expansion of human hematopoietic cells. However, when PDTX cells were treated with targeting compounds they all underwent massive apoptosis. Conversely, individual PDTX T-ALL could be selectively rescued by ECs, allowing the construction of individual drug response profile. To extend these data, 7 PDX T-ALL samples were screened against a 430-targeted compound library in supplemented RPMI or Stem Span media. Results indicated differential cell killing and gain (NFKB, BTK) and loss (TP-53, IGF-1R) of targets. Conclusions. These data clearly demonstrate a key role of aberrantly activated vascular niche in T-ALL cell maintenance and drug resistance. We envisage that drug screening of EC+T-ALL will lead to the identification of actionable targets in each individual patient. Our report supports the potential for future personalized curative strategies aimed at targeting both tumor cells and host tissue supporting niche elements disrupting pro-tumorigenic signals within leukemia cell niches. Disclosures Foà: Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy; Pfizer: Speakers Bureau; Ariad: Speakers Bureau. Rafii:Angiocrine Bioscience: Equity Ownership, Other: Non-paid consultant.

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Development of a Novel Phthalimide Derivative, Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Blood ◽

10.1182/blood.v124.21.5718.5718 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5718-5718

Author(s):

Yutaka Hattori ◽

Maiko Matsushita ◽

Noriko Tabata ◽

Hirokazu Shiheido ◽

Hiroshi Yanagawa ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Cell Lines ◽

Pharmacokinetic Study ◽

Bone Disease ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Phthalimide Derivative

Abstract BACKGROUND: Despite recent advances in the use of newly developed drugs including immune-modulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide and proteasome inhibitors such as bortezomib, carfilzomib, and MLN9708, MM is still an incurable disease. In particular, MM patients harboring 17p deletion, t(14;16), t(14;20), or t(4;14) are classified as a high-risk group and have shown significantly shorter survival. With the goal of helping prolong the survival of these high-risk MM patients, we screened 29 synthetic phthalimide derivatives and found a novel compound, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), which induced the apoptosis of KMS34 cells with t(4;14) and del17p13. PURPOSE:The purpose of this project is to clarify preclinical effects of the synthetic phthalimide derivative, TC11, on high-risk MM cell lines and osteoclasts. Namely, anti-myeloma and anti-osteoclastogenic activities and pharmacokinetic study in mice were shown. We also try to isolate directly binding molecules. Safety issues including hematological toxicities and teratogenicity were also discussed. METHODS AND RESULTS: TC11 significantly inhibited growth of MM cell lines (IC50 4-8μM) including KMS34 and KMS11 cells which have high-risk chromosomal abnormalities. TC11 also suppressed the proliferation of all of the bone marrow cells obtained from the MM patients, in a dose-dependent manner. TC11 increased annexin V-positive fraction and induced apoptosis. TC11 was injected intraperitonealy into myeloma (KMS34 and KMS11 cells)-bearing lcr/SCID mice, and anti-myeloma activity was evaluated in vivo. Twenty mg/kg of TC11 significantly inhibited growth of KMS34 or KMS11-derived plasmacytomas. Apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not significantly suppressed, suggesting low hematopoietic toxicity. In the pharmacokinetic analyses using lcr mice, the plasma concentrations of TC11 was examined; Cmaxwas 18.1μM at 1.5hr (Tmax), and T1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, Cmaxwas 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr. Oral administration of TC11 to Icr mice was safely carried out, and results of pharmacokinetic study will be shown. Aiming at the therapeutic use of TC11 to bone disease, anti-osteoclastogenic activity was examined. Mouse bone marrow mononuclear cells were incubated in the presence of M-CSF and RANK-ligand. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in number in the presence of 1μM of TC11. It was also found that 1μM of TC11 inhibited bone resorption by pit assay. We have identified nucleophosmin 1 (NPM1) and α-tubulin as TC11-binding molecules using our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. However, cereblon (CRBN) was not detected as a TC11-binding protein by this method. The immunofluorescent analysis showed that TC11-treated cells exhibited elevated levels of α-tubulin fragmentation. Together with our previous observation of induction of centrosomal disruption of HeLa cells by NPM1-knock down, TC11 may cause anti-myeloma effects via mitotic catastrophe. CONCLUSION: We have demonstrated that TC11, a novel phthalimide derivative, has anti-tumor activity against MM cells with high-risk genetic abnormality including del 17p and t(4;14), in vitro and in vivo. This novel compound also down-regulates the differentiation and function of osteoclasts. Our data provide a strong preclinical rationale for TC11 as a safe and effective drug for the treatment of high-risk MM patients with bone disease. The actions of this drug relating to α-tubulin and NPM1 remain to be further investigated. TC11 exerts its anti-myeloma effect via molecular interactions which do not involve CRBN. In addition, TC11 does not form racemate and is expected to lack teratogenicity. The results of our present study suggest that new phthalimide derivatives other than thalidomide, lenalidomide and pomalidomide could be developed by drug designing for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

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Carbenoxolone Decreases the Microenvironment-Induced Chemoresistance of Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood-2018-99-118956 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1474-1474

Author(s):

Farah Kouzi ◽

Frederic Picou ◽

Jerome Bourgeais ◽

Nathalie Gallay ◽

Fabrice Gouilleux ◽

...

Keyword(s):

Bone Marrow ◽

Gap Junctions ◽

Gap Junction ◽

Cell Lines ◽

Culture System ◽

Myeloid Leukemia ◽

Connexin 43 ◽

Leukemic Cells ◽

Leukemia Cells ◽

Hematopoietic Stem

Abstract Introduction Bone marrow niche of acute myeloid leukemia (AML) is a highly specialized microenvironment that regulates leukemic progression by favoring leukemic cell quiescence and chemoresistance. Niche components, especially mesenchymal stromal/stem cells (MSCs), have pro-survival effects on leukemia cells by protecting them from chemotherapy-induced apoptosis. The protection mechanisms of AML cells by the microenvironment are extensively studied to identify pharmacological targets to prevent AML relapse after chemotherapy. Direct intercellular communications between hematopoietic stem cells and MSCs involve connexins, such as connexin 43 (Taniguchi Ishikawa E et al. Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells. Proc Natl Acad Sci U S A. 2012,109:9071-6). The connexins are key components of gap junctions and we postulated in this study that blocking their assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. To this end we evaluated the effects of carbenoxolone (CBX), a glycyrrhetinic acid derivative known to block gap junctions (Rozental R et al. How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 2001;154:447-76), already evaluated in the treatment of human diseases. Methods Primary AML blasts isolated from bone marrow patients and 8 AML cell lines (KG1-a, KG1, HL-60, THP-1, MOLM-13, MV4-11, ML2, NB4) were exposed to CBX to assess cell viability and proliferation (Trypan blue exclusion and MTT assays). To study the combined drug effects of CBX and cytarabine (Ara-C), isobolograms were determined by co-treatment of AML cell lines with various doses of these two molecules. The metabolic effects of CBX on AML live cells were investigated with Seahorse® analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and high-throughput metabolic profiling was performed by OmniLog® technology. Direct cell-cell interactions were studied in a co-culture system of AML cells and bone marrow primary MSCs combining or not CBX (150 µM) and Ara-C (1 µM). After 48h (37°C, 5% CO2), AML cells adherent to MSCs were collected and the two populations were discriminated by flow cytometry studies (AML cells: CD45+CD90- / MSCs: CD45-CD90+) to quantify cell number apoptosis/necrosis (AnnexinV/7AAD assay). Gap junction inhibition by CBX was checked by calcein transfer (dye transferred mainly by gap junction channels) from labeled MSCs to AML cells in the co-culture system. Statistical analyses were performed with Mann-Whitney-Wilcoxon test. Results Treatment of AML cell lines with CBX reduced cell growth and viability in a time- and dose-dependent manner and the CBX IC50 was around 150 µM. Moreover, the exposure for 48h of primary AML blast to this dose of CBX induced a two time decrease in the number of viable cells. Interestingly, the isobolograms of the 8 AML cells lines identified three different profiles of resistance to chemotherapy and a synergistic effect between CBX and Ara-C. Regarding the energy metabolism, the analysis of all AML cell lines showed a major reduction of OCR and ECAR after treatment with CBX (150 µM, 48h) regardless their chemoresistance to Ara-C. Moreover, treated AML cells lacked their metabolic capacities for utilization of numerous substrates. In the co-culture experiments, contact with MSCs induced resistance to Ara-C; the apoptosis/necrosis rate observed after Ara-C exposure was 4.3- and 6.4-times decreased by MSC-contact for KG1a cells (n=5, p=0.0115) and primary blasts (n=5, p=0.0001), respectively. Co-treatment of leukemic cells with CBX and Ara-C reversed these deleterious effects of MSC-contact; the apoptosis/necrosis rate observed in MSC-contact leukemic cells was 5- and 2-times increased by CBX co-treatment for KG1a cells (n=5, p=0.007) and primary blasts (n=5, p=0.042), respectively. Finally, CBX induced a 48%-decrease in calcein transfer between leukemic cells and MSCs, highlighting its role as a gap junction inhibitor. Conclusion Niche-induced chemoresistance is associated with the mechanism of AML relapse after initial well-conducted chemotherapy. Combined to chemotherapy, CBX could be of interest to reduce the deleterious effects of leukemic niche by targeting gap junctions. Disclosures No relevant conflicts of interest to declare.

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Demonstration of Rapid Functional Myeloid Cell Recovery after Infusion of a Universal Donor Ex Vivo Expanded Progenitor Cell Therapy As a Bridging Graft Source

Blood ◽

10.1182/blood.v128.22.3348.3348 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3348-3348

Author(s):

Fabiola V. Merriam ◽

Suzan Imren ◽

Robert A Landeros ◽

Colleen Delaney

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

De Novo ◽

Ex Vivo ◽

Myeloid Cells ◽

Nsg Mice ◽

Donor Graft ◽

Universal Donor

Abstract Cord blood transplant (CBT) recipients are known to be at risk for delayed engraftment, resulting in an increased risk of morbidity and mortality post transplant. To overcome delayed engraftment, several groups have developed methods to expand ex vivo cord blood stem/progenitor cells (HSPC) which are under clinical evaluation. The majority of these expansion methods require identification of a patient specific cord blood donor as the source material for expansion, resulting in delays in the time to transplant and inherently carry a risk of product failure. In contrast, we have developed an off-the-shelf, universal donor ex vivo expanded cord blood (CB) derived HSPC product intended for use as a transient graft source which has been demonstrated to significantly reduce the incidence of documented bacterial infections in both transplant and non-transplant settings.1,2 Donor chimerism studies conducted weekly in the first month post transplant confirm that the initial early (days 0-14) myelomonocytic engraftment is derived largely from our universal donor graft. Herein, we now demonstrate that the these rapidly engrafting myelomonocytic cells generated from the universal donor graft source are mature and functionally intact human myeloid cells that can fight infectious organisms. CBT recipients enrolled on a phase II myeloablative CBT trial were included in these ancillary studies in which we evaluated the functional capacity of newly generated myeloid cells in peripheral blood. A flow cytometry-based assay which allowed quantitation of both phagocytosis and O2-dependent killing (oxidative burst) in myeloid cells was used. Strikingly, both monocytes (CD14+) and granulocytes (CD15+) in patients' blood displayed similar frequencies of phagocytosis and O2-dependent killing of Staphlococcus aureus at day 7 (90.3%±2.2% phagocytosis and 88.9±5.2% O2-dependent killing n=2) when more than 95% of myeloid cells were from the expanded cell product compared to day 14 (69±13.2% phagocytosis and 94±2% O2-dependent killing, n=2) when more than 99% of cells were from a non-manipulated CB unit as a result of immunologic rejection by the T cell replete CB unit. These findings provide strong evidence that de novo generated myeloid cells from expanded HSPCs are as functionally competent as myeloid cells de novo generated from non-expanded CB. To better study the functionional properties of myeloid cells derived in vivo from rapidly repopulating expanded CB HSPCs, we transplanted either 20,000 non-expanded (NE-HSPC) CD34+ CB cells or their expanded progeny (E-HSPC) into sub-lethally irradiated NOD-scid IL2rγnull (NSG) mice. At day 7 after transplantation mice transplanted with E-HSPC showed 40-fold higher human engraftment in the bone marrow than mice transplanted with NE-HSPC (28.3 ± 1% vs 0.7±0.1%, n=3, p<0.001). Remarkably, the monocytes and granulocytes from their bone marrow showed a similar phagocytic potential to that of the monocytes and granulocytes of mice receiving NE-HSPC (60.4±3.2% vs 69.6±3.2%, n=3, p=0.06). Moreover, the frequency of phagocytosis in the myeloid cells isolated from the lungs of mice receiving E-HSPC was 7-fold higher than in the lungs of mice receiving NE-HSPC. It has been well documented that E-HSPC when infused alone, also contribute to long term engraftment in NSG mice, and therefore at 22 weeks after transplantation, the frequency of phagocytosis in monocytes and granulocytes isolated from the bone marrow of mice receiving E-HSPC remained similar to that in the bone marrow of mice receiving NE-HSPC for Staphlococcus aureus (55.1 ±1.9% vs 43.8%±7%, n=5, p=0.15), Escherichia coli (50.8±2% vs 49 ±8.3%, n=5, p=0.83) and Zymosan (43.7%±3 vs 49.9%±9.2%, n=5, p=0.54) indicating the continued generation of functional myeloid cells from long term repopulating cells. We demonstrate for the first time that ex vivo expanded CB HSPCs rapidly give rise to functional myelomonocytic cells in vivo in patients and immunodeficient mice. This study validates that our universal donor off-the-shelf, expanded CB HSPC cell product is a valuable resource for patients undergoing myeloablative CBT, and further warrants its widespread use in a non-transplant setting as a supportive "myeloid bridge" to mitigate treatment-related morbidity and mortality. 1. Delaney C. et al. Lancet Haematol. 2016 Jul;3(7):e330-9 2. Summers C. et al. Blood 2014 124:3860 Disclosures Delaney: Nohla Therapeutics: Employment.

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Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.1648.1648 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1648-1648 ◽

Cited By ~ 1

Author(s):

Yaser Heshmati ◽

Gözde Turköz ◽

Aditya Harisankar ◽

Sten Linnarsson ◽

Marios Dimitriou ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Therapeutic Target ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Loss Of Function ◽

Potential Therapeutic Target ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

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Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽

10.1182/blood.v120.21.4295.4295 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4295-4295

Author(s):

Jae-Hung Shieh ◽

Tsann-Long Su ◽

Jason Shieh ◽

Malcolm A.S. Moore

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Culture System ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Cd34 Cells ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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Modeling Growth Of Pediatric T-ALL In Vivo and In Vitro: Clinical Meaning and Activation Of The NFkB Pathway

Blood ◽

10.1182/blood.v122.21.2571.2571 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2571-2571

Author(s):

Sandrine Poglio ◽

Xavier Cahu ◽

Benjamin Uzan ◽

Hélène Lapillonne ◽

Thierry Leblanc ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Stromal Cells ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Nsg Mice ◽

Nfkb Activation ◽

Two Systems

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the proliferation of T-cell precursors in various sites, such as thymus, bone marrow, blood, lymph nodes or central nervous system. As T-ALL cells alone do not successfully grow in vitro, xenografts of T-ALL cells into NOD/scid/IL-2R null (NSG) mice and long-term co-cultures of T-ALL cells with stromal cells have been developed to study the biology of T-ALL cells (Armstrong et al, Blood, 2009). However, the growth of T-ALL cells in these two systems is highly variable across T-ALL samples. Moreover, the clinical relevance of both assays and, except for NOTCH pathway activation, the molecular pathways involved in successful in vivo and in vitro growths are still elusive. The aim of this work was to determine the relationships between clinical, biological and molecular characteristics of human T-ALL at diagnosis and the growth of T-ALL in these two systems. Human T-ALL blood samples were collected at diagnosis from pediatric or young adult patients with T-ALL. 50,000 T-ALL cells were intravenously injected into NSG mice. Mouse bone marrow samples were collected every 3-4 weeks from day 35 to day 210 post-transplant. Leukemic engraftment was monitored using flow cytometry measuring the % of human CD45+CD7+ leukemic cells. Time to leukemic engraftment (TTL) was defined as the time between T-ALL injection and the detection of ≥20% leukemic cells in at least one mouse. In vitro co-culture growth assay consisted in plating 200,000 cells on MS5 or MS5-DL1 (Armstrong, Blood, 2009) and count every 7 days up to 28 days. A total of 36 samples were tested of which 22 (61%) engrafted into mice. Global median TTL was 82 days (range, 36-121) defining short (TTL<82 days) and long or no engraftment (TTL>82 days) TTL groups. Patient gender, age, mediastinal involvement or abnormal karyotype had no significant impact on TTL. A trend for a shorter TTL was observed for T-ALL samples with a white blood cell count (WBC) > median WBC = 146 G/L (p =0.06). Samples containing more than 20% of TCRαβ or CD8 positive cells exhibited increased incidence of engraftment (p = 0.049 and p=0.04 respectively) whereas CD34, CD1a, CD4 or sCD3 markers were not significantly correlated with TTL. Unlike samples with TLX1, TLX3 overexpression or NOTCH/FBXW7 mutations, samples with SIL-TAL1 deletion exhibited a shorter TTL (p = 0.0004). The 2-year progression free survival of “short TTL” patients was 72% vs 70% for patients with “longer TTL” or no engraftment (p=0.38). T-ALL samples for which growth could be achieved on MS5 cells also displayed a shorter TTL. To unravel molecular mechanisms involved in the growth of leukemic cells in these two systems, micro-arrays were performed for 8 “short TTL” T-ALL versus 8 “long TTL or no engraftment” T-ALL. 346 genes were differentially express in short TTL samples compared to long/no TTL samples (P<0.05, fold change: 1.5). As expected, most of genes up-regulated in short TTL group were implicated in cell cycle function enhancing the commitment of cells to S/M phases. Analysis of regulated networks revealed that several indirect modulators of NFkB (MAL, AhR and CYLD) were significantly up/down regulated in short TTL patient samples resulting in NFkB activation. Overall, T-ALL with SIL-TAL1 deletion display an increased ability to engraft into NSG mice, in accordance with increased WBC in T-ALL patients. Contrary to B-ALL, shorter TTL is not associated with poor prognosis in T-ALL. Moreover, NSG engraftment and co-culture on stromal cells are well correlated. A shorter TTL seems to be associated with an increased leukemic proliferation through NFkB activation. Disclosures: No relevant conflicts of interest to declare.

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