ALBERT

Description: The majority of long-term reconstituting hematopoietic stem cells (LT-HSCs) in the adult is in G0, whereas a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSCs compared with short-term reconstituting HSCs and progenitors and that SCL negatively regulates the G0-G1 transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at approximately 1 competitive repopulating unit was 2-fold decreased when Scl gene dosage was decreased. Second, Scl+/− HSCs were at a marked competitive disadvantage with Scl+/+ cells when transplanted at 4 competitive repopulating units equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared with controls. At the molecular level, we found that SCL occupies the Cdkn1a and Id1 loci in primary hematopoietic cells and that the expression levels of these 2 regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence.

Description: During the last 50 years, with over a million transplants, hematopoietic stem cell (HSC) transplantation has been the first and most extensively exploited stem cell therapy and the only curative regime for most acute leukemias. Nevertheless, the success of HSC transplantation and all other intensive ablative chemotherapy regimes is still overshadowed by a procedure-associated mortality of ~25%, due to both graft versus host disease and to the infectious complications associated with the severe leucopenia following bone marrow (BM) ablation, even in spite of standard support with HSC-stimulating factor G-CSF. It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response can severely compromise HSC regeneration. Here we show that the NAD+-boosting agents Nicotinamide Riboside (NR) and Nicotinamide MonoNucleotide (NMN) reduce mitochondrial activity within HSCs through increased mitochondrial clearance via autophagy and possibly the Unfolded Protein Response mitochondria (UPRmt), leading to increased asymmetric HSC divisions as measured by asymmetric mitochondrial distribution in single cell pair-daughter analysis. This process was abrogated in Nrk1-/-;Nrk2-/- double knock out mice, which cannot incorporate NR into the NAD+ salvage pathway. Contrary to controls, purified murine HSC underwent self-renewal in minimal culture conditions in presence of NR, and human CD34+ hematopoietic progenitors (hCD34+) cultured in vitro in presence of NR could repopulated NSG mice in primary and secondary transplant recipients. In vivo, NR dietary supplementation resulted in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improved survival by 80%, and accelerated blood recovery after murine lethal irradiation and limiting-HSC transplantation. In human xenotransplanted immune-deficient NSG mice, NR increased the production of human leucocyte progeny from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD+ boosting strategies on the most primitive blood stem cells, establishes a novel link between mitochondrial stress, mitophagy and stem cell fate decision, and unveils the potential of NR in vivo supplementation to improve recovery of patients suffering from hematological failure including post-chemo/radiotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4749 Hematopoietic stem cell (HSC) transplantation is the first successful cellular therapy and remains the only treatment providing long-term cure in acute myeloblastic leukemia. At the apex of the hematopoietic system, quiescent HSCs are spared by chemotherapeutic treatments that target proliferating cells and therefore can regenerate the entire blood system of a patient after drug exposure. Nevertheless, the consequence of repeated chemotherapy regimen on HSC function remains to be clarified. We previously showed that Scl/Tal1 gene dosage regulates HSC quiescence and functions when transplanted at limiting dilutions (Lacombe et al., 2010). In the present study, we investigate how massive expansion in vivo influences stem cell functions. To address this question, we optimized a protocol based on 5-fluorouracil (5-FU), an antimetabolite that has been used to treat colon, rectum, and head and neck cancers. In addition, we used Scl+/− mice to address the role of Scl in controlling HSCs expansion post-5-FU. We show that within 7 days following 5-FU treatment, HSCs exit quiescence and enter the cell cycle. To deplete cycling HSCs, we injected a second dose of 5-FU and showed that the stem cell pool was disseminated. Nonetheless, the remaining HSCs proliferated extensively to re-establish the HSC pool, which was twice larger than that of untreated mice. At this point, most HSCs have exited the cell cycle and were back to quiescence. Despite a near normal stem cell pool size and a quiescent status, HSCs from these 5-FU treated mice could not compete against untreated cells to regenerate the host in transplantation assays. Furthermore, we show that this extensive proliferation in vivo severely impaired the clonal expansion of individual HSC as measured by the mean activity of stem cell (MAS). Our results demonstrate that HSCs lose their competitive potential after two 5-FU treatments, suggesting that HSCs have an intrinsic expansion limit beyond which their regenerative potential is impaired. In addition, Scl is haplodeficient for cell cycle entry and cell division but Scl gene dosage does not affect this expansion limit. Therefore, our data dissociate the control of HSC expansion under extensive proliferative stress from cell cycle control during steady state. We surmise that chemotherapy regimen based on repeated administration of 5-FU or other antimetabolites are likely to severely impair long-term stem cell functions. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 207 Normal thymic progenitors are devoid of self-renewal capacity, which is a distinctive stem cell property. These thymic progenitors progress into the thymus through several stages of differentiation (DN1, DN2-4, DP) before giving rise to CD4+ or CD8+ immunocompetent cells that are released into the periphery. Therfore, thymic output requires continuous seeding from stem cell-derived progenitors. T cell acute lymphoblastic leukemia (T-ALL) is a common cancer in children. Almost 25% of childhood T-ALL involve the SCL transcription factor and/or its nuclear partners LMO1/2 and more than 50% harbour gain of function mutations of NOTCH1. Using a transgenic mouse model that reproduces the human disease, we previously showed that activation of SCL, LMO1 and Notch1 in the thymus is sufficient to transform thymocyte progenitors and induce T-ALL (Tremblay M et al., 2010). Here, we explore the mechanism of transformation by these three oncogenes in primary thymocytes during the pre-leukemic stage. Our results indicate that the SCL and LMO1 oncogenes collaborate to confer an aberrant self-renewal potential to a subset of pre-leukemic thymocytes, via induction of a stem cell gene signature that also distinguishes primary T-ALL patient samples in which LMO2 is expressed. Furthermore, our clonality and functional analyses indicate that only a few clones of preleukemic thymocytes from Scl-Lmo1 mice are able to colonize the thymus of recipient mice in transplantation assays and produce mature T-cells during the pre-leukemic stage. Precisely, we show that self-renewal activity is enriched in DN3 thymocytes which eventually acquire Notch1 gain of function mutations and leukemia initiating activity. On the other hand, the Notch1 oncogene by itself does not confer self-renewal properties to thymocytes. Rather, we show that the Notch1 oncogene enhances the activity of the SCL-LMO1 oncogene to increase the frequency of pre-Leukemic Stem Cells (pre-LSCs) without modifying the clonal expansion of individual pre-LSC when transplanted at limiting dilutions. These results indicate that the Notch1 oncogene modifies the self-renewal activity enforced by the SCLtgLMO1tg oncogenes into a self-renewal of expansion, typical of a transformed state. Furthermore, NOTCH1 confers an invasive potential to SCLtgLMO1tg thymocytes that become thymus-independent and acquire the capacity to develop in peripheral organs. Therefore, our observations are consistent with the view that the SCL-LMO1 oncogenic transcription factors reprogram DN3 thymocytes to acquire self-renewal potential, thereby establishing a pre-leukemic state. Finally, NOTCH1 activation provides a strong signal that collaborates with the SCL-LMO1 oncogenes to induce T-ALL by favoring self-renewal divisions in pre-LSC together with an invasive capacity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.

Description: Alterations of the BM microenvironment have been shown to occur after chemoradiotherapy, during aging, and after genetic manipulations of telomere length. Nevertheless, whether BM stromal cells adopt senescent features in response to these events is unknown. In the present study, we provide evidence that exposure to ionizing radiation (IR) leads murine stromal BM cells to express senescence markers, namely senescence-associated β-galactosidase and increased p16INK4a/p19ARF expression. Long (8 weeks) after exposure of mice to IR, we observed a reduction in the number of stromal cells derived from BM aspirates, an effect that we found to be absent in irradiated Ink4a/arf-knockout mice and to be mostly independent of the CFU potential of the stroma. Such a reduction in the number of BM stromal cells was specific, because stromal cells isolated from collagenase-treated bones were not reduced after IR. Surprisingly, we found that exposure to IR leads to a cellular nonautonomous and Ink4a/arf-dependent effect on lymphopoiesis. Overall, our results reveal the distinct sensitivity of BM stromal cell populations to IR and suggest that long-term residual damage to the BM microenvironment can influence hematopoiesis in an Ink4a/arf-dependent manner.

Description: The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p