Acute myeloid leukemias (AML) are aggressive blood cancers characterized by an overall survival of 27% at 5 years. The main challenge in AML treatment originates from the genetic heterogeneity of the ...disease that contributes to the wide range of clinical outcomes observed. A large proportion (~35%) of AML patients exhibit no distinguishable chromosomal abnormalities that can be used to guide treatment selection and are therefore classified in the poorly characterized intermediate risk category. Approximately 50-60% of intermediate risk AML patients carry mutations in the NPM1 gene. These mutations are associated with a favorable outcome unless a concomitant mutation in the FLT3 gene is detected, which accounts for 39% of cases. The survival rate is further worsened when a third mutation is detected in the DNMT3A gene, dropping from 40% to 20% 5 years post-treatment for double and triple mutants, respectively.
This study aimed to identify drugs selectively affecting the viability of leukemic cells from AML patients with NPM1 mutations. To achieve this, we took advantage of culture conditions developed by our group that prevent differentiation of leukemic cells and preserve leukemia stem cell activity from primary AML specimens (Pabst et al., Nature methods, 2014), enabling chemical screening of primary AML specimens. We conducted a chemical screen using a collection of ~300 clinical grade drugs on a cohort of 38 primary human AML specimens containing NPM1 mutated (NPM1c+) and NPM1 wild-type (NPM1wt) samples. These specimens belonged to the Leucegene collection of sequenced and clinically annotated samples. The screen identified ABT-199 as the compound with the most discriminatory effect toward NPM1c+ AML. ABT-199 is a specific BH3-mimetic that prevents anti-apoptotic BCL2 from binding pro-apoptotic BAX and BAK1 proteins, leading to apoptosis. ABT-199 demonstrated encouraging results for AML treatment, but the determinants of drug sensitivity have not been well defined. Analysis of the enrichment of clinical variables in ABT-199 sensitive and resistant groups of AML specimens to define characteristics/biomarkers associated with ABT-199 sensitivity in AML revealed that mutations in NPM1, RAD21, IDH1, IDH2, DNMT3A and FLT3 (ITD), as well as normal karyotype and the FAB M1 class all significantly associate with increased ABT-199 sensitivity. At the other side of the spectrum, mutations in TP53 and FAB class M5B were significantly enriched in the resistant group. Additional analyses revealed that NPM1c+/DNMT3Amut/FLT3-ITD specimens are sensitive to ABT-199, which may provide a rationale to prioritize patients from this adverse risk AML subgroup for explorative ABT-199 based regimens. Specimens with RAD21 mutations were the most sensitive to ABT-199 treatment and further analyses demonstrated a clear association between mutation of cohesin genes (RAD21, SMC1A, SMC3, STAG2) and increased ABT-199 sensitivity. In line with this, we demonstrated that RAD21 knockdown alone is able to sensitise AML cell lines to BCL2 inhibition. Comparative transcriptome analysis of ABT-199 sensitive and resistant specimens also revealed an apoptotic gene signature linked to ABT-199 resistance with BCL2A1, an anti-apoptotic BCL2 homolog, being the most differentially expressed apoptotic gene between these response groups and showing increased expression in the resistant subset. Expression correlation analysis over the 415 specimens of the Leucegene cohort showed that BCL2A1 is one of the top genes anti-correlated to BCL2, and accordingly, high BCL2 and BCL2A1 expressors were enriched among ABT-199 sensitive and resistant specimens, respectively.
In conclusion, using an unbiased pharmacogenomic approach, we identified ABT-199, a compound with the potential to eradicate NPM1c+ AML, which has already been tested in a phase 2 clinical trial for AML. Our results shed light on determinants of ABT-199 sensitivity which could readily impact AML therapy by providing a rationale for prioritizing patients with NPM1, RAD21, IDH1 and/or IDH2 mutations for ABT-199 AML trials. Our results also uncover potential mechanisms of resistance to ABT-199, providing grounds to design combination therapies to overcome ABT-199 chemoresistance.
Sauvageau:ExCellThera: Employment, Equity Ownership.
60% to 70% of Acute Myeloid Leukemia (AML) patients enter complete remission after induction regimen, but the majority relapse within 3 years due to the outgrowth of therapy resistant Leukemia Stem ...Cells (LSCs). Identification of novel treatment strategies effective against these cells thus represents an outstanding medical need. We developed a cell culture method, which transiently maintains LSC activity ex vivo (Pabst et al., Nature Methods, 2014) and enables chemical interrogation of cell types relevant for the progression of the disease. Overall, HSCs and LSCs share numerous biological traits, making specific LSC eradication challenging. However, striking differences in energy metabolism between normal and leukemic stem cells have recently been suggested. While HSCs appear to rely primarily on anaerobic glycolysis for energy production, LSCs seem to depend on mitochondrial oxidative phosphorylation for their survival. Targeting mitochondrial respiration could therefore represent an effective approach for the specific eradication of LSCs.
We aimed to identify novel therapeutic targets for AMLs with poor treatment outcome. The study relied on the Leucegene approach that integrates results generated by RNA sequencing analysis of primary human AML specimens, detailed clinical and cytogenetic annotations provided by the Quebec leukemia cell bank and ex vivo responses of primary AML samples to various chemical compounds. Our study specifically focused on specimens originating from patients with poor (overall survival < 3 years) and good (overall survival ≥ 3 years) response to standard chemotherapy, and did not include cases of Acute Promyelocytic Leukemia (APL).
We identified Mubritinib, previously described as an ERBB2 inhibitor, as a novel anti-leukemic agent, which selectively inhibits the viability of leukemic cells from therapy-resistant AML patients, but does not affect normal CD34+ cord blood cells. Exposure to Mubritinib triggered apoptotic cell death in a subset of AML samples with high mitochondrial function-related gene expression, high relapse rates, and short overall survival. Sensitivity to Mubritinib also strongly associated with the intermediate cytogenetic risk category, normal karyotype (NK), and NPM1, FLT3 (ITD) and DNMT3A mutations. Conversely, resistance to Mubritinib associated with favorable cytogenetic risk AMLs, Core Binging Factor (CBF) leukemias and KIT mutations. Mubritinib has been developed as an ERBB2 kinase inhibitor. Intriguingly, we found that ERBB2 is not expressed in Mubritinib-sensitive AML specimens, suggesting that the anti-leukemic activity of this compound is likely not mediated by ERBB2 inhibition. Using a combination of functional genomics and biochemical analyses, we demonstrated that Mubritinib directly inhibits the mitochondrial Electron Transport Chain (ETC) complex I, which leads to a decrease in oxidative phosphorylation activity and to induction of oxidative stress. The impact of Mubritinib on AML progression was explored using a syngeneic mouse model (MLL-AF9 tdTomato-positive leukemia). Recipients of MLL-AF9 cells treated with Mubritinib exhibited a 19-fold decrease in the number of tdTomato-positive cells in the bone marrow and a 42-fold decrease in the spleens compared to control mice. Short-term treatment also led to a 37% increase in the median overall survival of Mubritinib exposed recipients compared to vehicle treated mice. Importantly, and in agreement with our observation that Mubritinib treatment does not impede proliferation of normal hematopoietic CD34+ cells in vitro, Mubritinib treatment had no impact on the number of non-transduced (tdTomato negative) nucleated bone marrow cells of recipients.
We uncovered the clinical, mutational, and transcriptional landscape of mitochondrial vulnerability in AML and identified Mubritinib as a novel ETC complex I inhibitor with therapeutic potential for approximately 30% of AML cases currently lacking effective treatment options. As Mubritinib completed a phase I clinical trial in the context of ERBB2-positive solid tumors, our work suggests an opportunity to re-purpose Mubritinib's usage for this genetically distinct subgroup of poor outcome AML patients.
No relevant conflicts of interest to declare.
Hoxa9, Meis1 and Pbx1 encode homeodomaincontaining proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with ...each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. In this study, we sought to determine if Hoxa9 could transform hemopoietic cells in collaboration with either Pbx1 or Meis1. Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induced growth factor‐dependent oligoclonal acute myeloid leukemia in <3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b failed to transform these cells acutely within 6 months post‐transplantation. Similar results were obtained when FDC‐P1 cells, engineered to overexpress these genes, were transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA‐binding partners in transformation of hemopoietic cells.
It is believed that hemopoietic stem cells (HSC), which colonize the fetal liver (FL) rapidly, expand to establish a supply of HSCs adequate for maintenance of hemopoiesis throughout life. ...Accordingly, FL HSCs are actively cycling as opposed to their predominantly quiescent bone marrow counterparts, suggesting that the FL microenvironment provides unique signals that support HSC proliferation and self-renewal. We now report the generation and characterization of mice with a mutant allele of Baf250a lacking exons 2 and 3. Baf250aE2E3/E2E3 mice are viable until E19.5, but do not survive beyond birth. Most interestingly, FL HSC numbers are markedly higher in these mice than in control littermates, thus raising the possibility that Baf250a determines the HSC pool size in vivo. Limit dilution experiments indicate that the activity of Baf250aE2E3/E2E3 HSC is equivalent to that of the wild-type counterparts. The Baf250aE2E3/E2E3 FL-derived stroma, in contrast, exhibits a hemopoiesis-supporting potential superior to the developmentally matched controls. To our knowledge, this demonstration is the first that a mechanism operating in a cell nonautonomous manner canexpand the pool size of the fetal HSC populations.
Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease ...initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal.
We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection.
To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker.
Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones.
In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment.
No relevant conflicts of interest to declare.
The three-amino-acid loop extension (TALE) class homeodomain proteins MEIS1 and PKNOX1 (PREP1) share the ability to interact with PBX and HOX family members and bind similar DNA sequences but appear ...to play opposing roles in tumor development. Elevated levels of MEIS1 accelerate development of HOX- and MLL-induced leukemias, and this pro-tumorigenic property has been associated with transcriptional activity of MEIS1. In contrast, reduction of PKNOX1 levels has been linked with cancer development despite the absence of an identifiable transactivating domain. In this report, we show that a chimeric protein generated by fusion of the MEIS1 C-terminal region encompassing the transactivating domain with the full-length PKNOX1 (PKNOX1-MC) acquired the ability to accelerate the onset of Hoxa9-induced leukemia in the mouse bone marrow transduction/transplantation model. Gene expression profiling of primary bone marrow cells transduced with Hoxa9 plus Meis1, or Hoxa9 plus Pknox1-MC revealed perturbations in overlapping functional gene subsets implicated in DNA packaging, chromosome organization, and in cell cycle regulation. Together, results presented in this report suggest that the C-terminal domain of MEIS1 confers to PKNOX1 an ectopic transactivating function that promotes leukemogenesis by regulating expression of genes involved in chromatin accessibility and cell cycle progression.
Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass with, however, undesirable long term consequences and remains ineffective in ...adolescent and adult T-ALL. Furthermore, relapse can be caused by pre-leukemic stem cells (pre-LSCs) that were spared by current protocols and evolved to malignancy. A distinctive characteristic of pre-LSCs is their critical dependence on interactions with the microenvironment for survival, which guided our strategy to target pre-LSCs using niche-based screening assays.
Using transgenic mouse models that closely reproduce the human disease, we showed that the SCL/TAL1 and LMO1 oncogenic transcription factors establish a pre-leukemic state by reprogramming normal pro-T cells into aberrantly self-renewing pre-LSCs (Gerby et al. PloS Genetics, 2014). We now provide direct evidence that pre-LSCs are much less chemosensitive than leukemic blasts to current drugs, due to a distinctive lower proliferative state as assessed by real-time imaging in a competitive assay. We therefore designed a robust protocol for high-throughput screening (HTS) of compounds targeting primary pre-LSCs that are maintained on stromal cells engineered for optimal NOTCH1 activation to mimick the thymic microenvironement. The multiparametric readout takes into account the intrinsic complexity of primary cells to specifically monitor pre-LSCs. We screened a targeted library of 1904 compounds and identified UM0119979 that disrupts both cell autonomous and non-cell autonomous pathways: UM0119979 abrogates pre-LSC viability and self-renewal activity in vivo by specifically inhibiting the translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remain functional. Moreover, in vivo administration of UM0119979 efficiently reduced the leukemia propagating activity of primary human T-ALL samples in xenografted mice. Finally, in addition to SCL-LMO-induced T-ALL, our results reveal a novel possibility of therapeutic intervention in MYC-dependent hematologic malignancies.
In summary, our screening assay, built on the genetic dependencies of pre-LSCs, revealed their vulnerabilities to compounds that inhibit both the primary oncogenes and non-cell autonomous pathways triggered by the microenvironment. The results illustrate how recapitulating tissue-like properties of primary cells in high throughput screening is a promising avenue for innovation in cancer chemotherapy.
No relevant conflicts of interest to declare.
Abstract 2474
Aberrant expression of Hox genes and their cofactors Pbx and Meis1 has been detected in approximately 50% of all human leukemias, and proteins interacting with these homeodomain factors ...could play a major role in leukemia development. Studies in drosophila showed that hth/MEIS directly interacts with YKI, a component of the Hippo signaling pathway (Peng HW et al., 2009). The core components of this pathway in the mammalian cells are the kinases MST 1 or 2 and LATS 1 or 2, and the downstream transcription cofactors WWTR1 and YAP (homologues of the drosophila Yki). The Hippo pathway has been proposed to play a tumor suppressive role in carcinoma development (Lu L et al. 2010), but little is known about its function in hematopoiesis and leukemia. To address this issue, we first determined the expression levels of the core Hippo pathway constituents in different subpopulations of primitive hematopoietic cells by quantitative RT-PCR. Hematopoietic stem cells (HSC) isolated from day 14.5 fetal liver (FL-HSC, phenotype: CD150+CD48-Lin-), or bone marrow from 3 and 4 week old mice (BM-HSC, phenotype: cKit+CD150+CD48-Lin-) express comparable levels of Lats 1/2 and Mst 1/2. FL-HSC, however, express approximately 3 fold higher levels of Wwtr1 and Yap than the BM-HSC. Expression of all core components of the Hippo pathway was also detected in the Hoxa9+Meis1-induced leukemia named FLA2 in which approximately 70% of cells represent leukemia stem cells (LSC). The role of this pathway in leukemia was assessed using the shRNA-mediated loss of function approach. For each core component, 5 different shRNAs were designed, and 2 achieving ≥40% decrease in the targeted transcript levels were selected for the in vivo experiments. Freshly isolated FLA2 leukemia cells were infected with recombinant retroviruses carrying the control shLuciferase or the targeting shRNA, and green fluorescent protein (GFP), and were transplanted into sub-lethally irradiated recipient mice. The proportions of shRNA transduced (GFP+) cells were determined at the time of transplantation (day 0), and at the time of sacrifice (day 18 ± 2). During this period, the proportions of shWwtr1(GFP+) cells to the leukemic cell populations decreased to 10–20% of the initial day 0 values. Conversely, the Lats1 knockdown leads to > 50% increase over the initial proportion of the GFP+ cells. The combined Lats1+Lats2 knockdown enhanced the competitiveness of the transduced cells compared shLuciferase controls. These significant results (p < 0.05, Mann-Whitney-Test) suggest that LATS kinases act as negative regulators of leukemic cell expansion. To exclude the possibility that this effect is limited to FLA2 leukemia we isolated the CD150+CD48-Lin- stem/progenitor cells from FL, co-infected them first with Hoxa9 and Meis1 cDNA carrying retroviruses, and then knocked down Wwtr1 or Lats1. Similar to observations in FLA2 leukemia model, Lats 1 depletion promoted ∼2-fold increase, and Wwtr 1 reduction >80% decrease in proportions of the transduced (GFP+) cells compared to their initial day 0 levels. Together, our observations suggest that LATS kinases act as negative modulators of Hox/Meis-induced leukemia and indicate a possibility for a specific targeting of the Hox/Meis-activated cellular pathways.
No relevant conflicts of interest to declare.
Abstract 1919
Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization ...regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC.
No relevant conflicts of interest to declare.
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