ALBERT

Beschreibung: While induction into remission is effective in the majority of acute myeloid leukemia (AML) patients, disease recurrence is common, especially among the elderly. Understanding the origins of AML relapse would permit better treatments targeting the specific cells that survive chemotherapy. While some evidence suggests that AML relapse can originate either from a minor or a major clone that is already present at diagnosis, the exact origins of AML relapse are still obscure. In the current study we aimed at identifying the origins of AML by identifying genetic variants that appear at relapse, and to then track these variants back into specific cell populations present at diagnosis. We hypothesized that relapse might have multiple origins: from the major blast population, from rare leukemia imitating cells (LIC) as detected using xenografting, or from preleukemic stem cells (preL-HSCs). Methods: The bulk diagnosis and relapse samples of peripheral blood from eleven AML patients were analyzed, first by whole genome sequencing (50X coverage) to identify somatic mutations and genetic variants which were specifically present at relapse (relapse variants-RVs). The presence RVs was then reassessed in phenotypically defined sub-fractions sorted from the diagnosis samples, at a sensitivity of 1 in 1000 by digital PCR. The following sub-populations were genotyped: 1) isolated CD33+ blasts (the major population) 2) phenotypically defined leukemic and preleukemic stem cells 3) functionally defined leukemia initiating cells (LICs) harvested from xenografts (an average of 30 xenografts were generated from each diagnosis and relapse sample). Results: LICs, but not the dominant blast population from diagnosis carried the RVs in 3 of 11 cases. In these patients CD33-CD34+CD45RA+ immature cells from diagnosis also carried the RVs. In a second subset of 3 of 11 AML samples, relapse originated from a minor clone present within the CD33+ leukemic blasts; these samples did not produce exnografts. Other samples (2/11) exhibited relapse samples that arose from a combined origin (both LICs, and CD33+ blasts, or from the major clone (1/11). In two cases we could not identify the origins of relapse. As our initial results suggested that the cells responsible for AML relapse can come from distinct origins within the diagnosis sample, we next asked whether other functional and phenotypic differences might be present between the patients that have different relapse origins. RNA sequencing analysis of bulk cells from diagnosis demonstrated a remarkable clustering of the global gene expression that correlated with the origin of relapse. Unsupervised hierarchical clustering grouped together the AML samples who relapsed from the LICs, while all other samples were in a very distinct second cluster. The gene expression signature of the samples that relapsed from LICs was consistent with a monocytic phenotypic signature, while the other samples were more progenitor-like. To further expand and validate our findings we used the same unsupervised clustering on the RNA sequencing data of AML samples who relapsed in the TCGA dataset (n=86). Remarkably, the similar two main clusters were generated; comparison by GSEA provided evidence that the gene expression clusters in our study were generated by the same genes as in the TCGA clusters. Conclusion: Our results provide for the first time evidence that AML can relapse from distinct, predictable and pre-existing origins: AMLs with a monocytic phenotype relapse from chemo-resistant LICs; and AMLs with a progenitor gene expression pattern (yet lacking xenografting capacity) that relapse from CD33+ cells. These results pose a series of predictions as to the success of different therapies. For example, in the former group the major monocytic clone is sensitive to chemotherapy, yet relapse originates from CD33-CD34+CD45RA+ cells and would therefore be predicted to be resistant to Anti-CD33 therapeutics. On the other hand, relapse in the latter group originates from CD33+ cells and these are predicted to be sensitive to Anti-CD33 therapeutics. The results of this study document the complexity in origins of AML relapse and have important implications for the design of future more effective and personalized strategies for preventing AML relapse. Disclosures No relevant conflicts of interest to declare.

Beschreibung: The NOD.SCID xenotransplantation assay is a key model system for interrogating the biology of leukemic stem cells (LSCs) in human acute myeloid leukemia (AML). Approximately 50% of AMLs can generate human grafts in immunodeficient mice that recapitulate the features of the parent sample. However, some AML samples generate non-leukemic grafts upon xenotransplantation. We recently reported that multilineage (ML) grafts, comprised of both B-lymphoid and myeloid cells, are generated by preleukemic hematopoietic stem cells (preL-HSCs; Shlush et al. Nature 2014). PreL-HSCs contain a subset of the mutations present in leukemic blasts and have a competitive growth advantage over wildtype HSCs leading to clonal expansion in vivo, yet retain multilineage differentiation capacity. To investigate the relationship between patient outcomes and the biological properties of LSCs and preL-HSCs as reflected by different engraftment patterns, we transplanted 272 diagnostic patient samples, representing a broad cross-section of adult AML, into sublethally irradiated NOD.SCID mice by intrafemoral injection. Human chimerism was assessed 8-10 weeks post-transplant by flow cytometry. 41% of samples generated AML xenografts, defined as a human graft containing 〉90% myeloid (CD33+CD19-CD45+) cells. Three patterns of engraftment were seen with the remaining samples: no human graft, defined as 10% CD19+CD33-CD45+ B-cells (22%). Among patients whose samples generated ML grafts compared to AML grafts or TC/no graft, secondary AML was less common (10% vs. 27% vs. 26%, respectively; P=0.03). Associations between engraftment pattern and other baseline clinical characteristics, including age, white blood cell (WBC) count and cytogenetics, did not reach statistical significance in this cohort. However, there was a strong correlation between AML engraftment capacity and response to standard induction chemotherapy. AML engrafters had lower complete remission (CR) rates compared to all other patients as a group (51% vs. 81%; P

Beschreibung: Background: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm (MPN) characterized by marked bone marrow fibrosis, extramedullary hematopoiesis and significant risk for leukemic transformation. Most patients carry recurrent MPN driver mutations in JAK2, CALR and MPL and many carry additional mutations in epigenetic regulators, splicing and signaling pathways. MF pathophysiology remains poorly understood, particularly with respect to the cellular identity of the MF clone(s) bearing mutations that initiate and maintain the disease. Therefore, here we tracked somatic mutations from nine MF patients in sorted hematopoietic cell populations from serial time-points as well as patient-derived xenografts (PDXs), to identify clinically relevant MF cell populations for future targeted therapies. Methods: Genomic DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of nine chronic-phase MF patients (5 JAK2+ and 4 CALR+) and sequenced using the 54-gene TruSight Myeloid targeted panel. PBMCs from the same patients at serial time-points (if available) were sorted into hematopoietic stem and progenitor cell (HSPC) and mature cell populations. In addition, PDXs were generated following injection of CD34+ peripheral blood cells into sub-lethally irradiated NSG and/or NSG-SGM3 mice. Human myeloid (CD45+33+) and B (CD45+19+) cells were isolated from PDXs after 8-16 weeks. Digital droplet PCR was used to interrogate known oncogenic variants, identified from the targeted sequencing, in all sorted primary and PDX cell fractions. Results: We reveal that the vast majority of genetic lesions used to interrogate the hierarchy were acquired at the level of immunophenotypically defined hematopoietic stem/multipotent progenitor cells (HSC/MPP) (CD45+34+38-RA-, termed MF-HSC). MF-HSC variants were generally shared with most HSPC, differentiated myeloid (CD33+) and lymphoid (B and/or T cell) populations sorted from primary patient samples. In our cohort, MPN driver mutations were invariably acquired in MF-HSC and consistently maintained over longitudinal clinical follow-up (median of 17 months) in both MF-HSCs and differentiated myeloid cells (CD33+). Interestingly however, in 2 cases (1 CALR+ and 1 JAK2+ MF), ASXL1 mutations in MF-HSCs were observed at the first time-point but restricted to the CD33+ compartment at a later time-point. Further, in one patient, SF3B1 mutation was restricted to the CD33+ fraction at the first time-point and was completely absent at the second time-point. Thus, certain mutations, like ASXL1, may be required for MF initiation but not necessarily disease maintenance; or alternatively, independent MF-HSC clones may outcompete others over time. Additionally, some acquired downstream mutations may be transient in nature and not clinically relevant. Together these data suggest that MF-HSCs are essential for MF clonal maintenance. To complement our genetic interrogation of the hematopoietic hierarchy in MF, we demonstrate that CD34+ HSPCs generate multi-lineage grafts in xenotransplanted mice bearing mutations identified in MF-HSCs. CALR+ samples invariably demonstrate multi-lineage potential and clonal dynamics in PDXs suggest that CALR mutations are likely the first genetic lesion acquired in multipotent MF-HSCs. JAK2+ samples generated more variable data, but by combining the genetic data of both PDXs and lymphoid cells sorted from primary patient samples, we show that JAK2+ MF-HSCs may also be multipotent. Taken together, these data suggest that MF-HSCs are multipotent, functionally relevant MF-initiating cells capable of propagating downstream HSPC populations and the CD33+ MF-disease clone that harbors all interrogated mutations. Thus, rare MF-HSCs are both disease-initiating and disease-maintaining cells. Conclusions: Our approach combining high-resolution cell sorting and xenograft assays with genomic interrogation demonstrates that MF is initiated and maintained by rare multipotent MF-HSCs. Our study underscores the complex evolutionary events that occur in the stem cell compartment during disease progression and identifies MF-HSCs as the relevant cellular target for future therapeutic intervention. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding.

Beschreibung: Acute myeloid leukemia (AML) patients with normal cytogenetics, NPM1 mutation, and no FLT3-ITD are considered to be at low molecular risk (LMR). We previously reported that most LMR patients have a low LSC17 score; these patients derive benefit from the addition of low fractionated doses of gemtuzumab ozogamicin (GO) to standard treatment and have favorable survival outcomes compared to patients with high LSC17 scores (Ng, Nature 2016). We recently developed a 13-gene sub-score (LMR13) that can be calculated from the LSC17 assay; a high LMR13 score identifies not only patients with molecularly-defined LMR disease, but also patients with LMR-like gene expression (GE), treatment response, and survival outcome. Similar to LMR cases, LMR-like patients gain a significant overall, event-free, and relapse-free survival (OS, EFS, RFS) benefit from the addition of GO treatment as observed in the ALFA-0701 trial cohort (OS: P=0.05; EFS: P=0.009; RFS: P=0.02). To gain mechanistic insight into the molecular determinants of GO response in LMR and LMR-like patients, we modelled the pathway through which GO traverses upon targeting a cell using a curated list of n=245 genes that includes the GO binding receptor CD33, lysosomal markers, ATP-binding cassette (ABC) transporters, DNA damage response/repair machinery, and pro/anti apoptotic factors. Sparse statistical regression was applied to a training dataset of n=495 AML patients (GSE6891) to identify the minimal subset of pathway components that were most associated with high LMR13 scores as a surrogate for GO responsiveness. This process selected n=16 genes which were then used to construct a random forest classifier to predict GO response. The final decision-tree based model, termed GO12, retained n=12 of the 16 pathway genes, and was able to accurately identify LMR and LMR-like patients across n=5 independent AML cohorts totaling n=1188 patients (AUC≈80.8%). As expected, ALFA-0701 patients who were predicted to be LMR-like with 〉50% certainty achieved significantly better survival when GO was added to their induction regimen (OS: P=0.03; EFS: P

Beschreibung: Acute myeloid leukemia (AML) is a devastating disease; however 20%-30% of patients will achieve long term remission. The long-term follow up of AML patients who received bone marrow transplantation (BMT) is well documented. It remains unclear though whether AML long-term survivors who did not undergo BMT (LTSnoBMT) return to normal health. In order to answer this question a large cohort of AML patients and well matched controls are needed. For the present study, the electronic health records (EHR) of 4.2 million individuals covering over 16 years of follow up were studied. Morbidity and mortality of LTSnoBMT were compared to a large number of age-matched controls. We identified 177 LTS who were in remission two years after AML diagnosis of which 61 were LTSnoBMT. The mortality of LTSnoBMT 10 years after diagnosis was 20%-50% higher in comparison to controls in an age dependent manner with higher mortality among the elderly LTS (Figure 1A, B). While several lab results were different between the LTSnoBMT and controls, most strikingly the red cell distribution width (RDW) of the LTSnoBMT was much higher among elderly LTSnoBMT (Figure 1C). High RDW has been previously reported to correlate with mortality. Recent studies found that high RDW predict AML and even greater correlation of high RDW with mortality among individuals carrying age related clonal hematopoiesis (ARCH) mutations was detected. To investigate the genetic profile of LTSnoBMT, a deep-targeted sequencing method (covering 32 recurrently mutated genes in AML) was used. Remission samples (at least 2 years from diagnosis) from 28 LTSnoBMT were analyzed. In most cases mutations which were detected at diagnosis, were not present in the remission samples. ARCH defining events (most of them new) were found in 64% of the LTSnoBMT. To evaluate whether this high prevalence of ARCH related mutations among LTSnoBMT patients is related to chemotherapy exposure, we also studied 30 individuals with lymphoid malignancies who received chemotherapy and were in remission. The prevalence of ARCH related mutations in the controls was significantly lower (37%) than in the LTSnoBMT (Figure 2A) (p=0.035). Higher prevalence of IDH1/2 mutations was found among LTSnoBMT (Figure 2C) (p=0.017). Furthermore, 70% of the mutations detected in the LTSnoBMT were recurrent (i.e. has more than 5 occurrences in COSMIC data base) compared to 31% in the controls (Figure 2B) (p=0.001). Altogether, AML is a complex disease where even after the sustainable eradication of the malignant clone, the hematopoietic system/microenvironment does not normalize. The reported correlation between ARCH and mortality suggests that the high prevalence of ARCH related mutations among LTS might contribute to the high mortality that we observed. Our findings have both biological and clinical implications. Future studies should determine the reasons why AML - LTS have such high prevalence of ARCH with specific mutations and increased mortality. Furthermore, closer clinical follow up including mutation analysis should be considered for these individuals. Disclosures Cilloni: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy. Ofran:Novartis: Other: Served on a Novartis advisory board.

Beschreibung: miRNA expression is deregulated in human acute myeloid leukemia (AML), however the impact of altered post-transcriptional programs on the genesis and maintenance of leukemia stem cells (LSC) remains undefined. In order to elucidate the functional role of miRNA in LSC and identify relevant miRNA candidates, we performed global miRNA profiling on sorted cell subpopulations from 16 AML patient and 3 umbilical cord blood samples (Eppert et al, Nature Medicine 2011). Supervised analysis guided by the ability of each sub-population to initiate leukemic engraftment after xenotransplantation into immune-deficient mice generated a unique miRNA signature. miR-126, a miRNA that we previously demonstrated to have a conserved role in maintaining hematopoietic stem cell (HSC) quiescence (Lechman et al. Cell Stem Cell, 2012), was more highly expressed in LSC-enriched fractions and chosen for further validation. To confirm that miR-126 is a bona fide LSC determinant, we utilized a bidirectional lentiviral reporter vector specific for miR-126 (Gentner et al. Science Translational Medicine, 2010) to sort cells from AML patient samples based on miR-126 bioactivity, and demonstrated that all in vivo leukemia-initiating capacity was confined to cells with elevated miR-126 bioactivity. Lentiviral enforced expression of miR-126 in primary AML patient samples significantly increased LSC frequency (3.5-52.3 fold) as assessed by limiting dilution transplantation assays, while diminishing cell cycle entry, differentiation marker expression (CD14,CD15) and colony forming potential. Sponge-mediated knockdown of miR-126 expression resulted in the opposite effects. These findings suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Moreover, by preserving LSC quiescence miR-126 promoted chemotherapy resistance, in part through suppression of CDK3, a gatekeeper of G0 to G1 cell cycle transit. Enforced expression of CDK3 partially rescued the functional consequences of supra-physiological levels of miR-126 bioactivity, rendering previously resistant LSC susceptible to killing by AraC/Daunorubicin combination chemotherapy. Our human LSC miRNA signature, optimized by regression analysis on a cytogenetically normal AML patient cohort, was prognostic for survival in a large independent AML patient cohort (Ley et. al N Engl. J Med, 2013) further validating the clinical significance of miRNA as stem cell determinants. Furthermore, miRNA-126 alone was prognostic for survival in two independent cohorts of AML patients with normal cytogenetics. These data demonstrate a mechanistic role for miR-126 in governing intrinsic LSC properties and establish miR-126 as a critical biomarker for clinical outcome. Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

Beschreibung: The NOD.SCID xenotransplantation assay has emerged as a key model system for interrogating the biology of human acute myeloid leukemia (AML). Previous work has established that approximately 50% of AMLs can generate grafts resembling the patient’s leukemia in immunodeficient mice. An understanding of the biologic properties of AML that enable engraftment in xenotransplant assays may aid in the development of prognostic tools and individualized therapy. To investigate these properties in a broad cross-section of human AML, we transplanted bone marrow (BM) or peripheral blood (PB) cells from 307 AML patients intra-femorally into sublethally irradiated NOD.SCID mice pre-treated with an anti-CD122 antibody. Human engraftment in the recipient BM was assessed 8-10 weeks post-transplant via flow cytometry. AML xenografts, defined as a human graft composed of 〉90% myeloid (CD33+CD19-CD45+) cells, were generated by 134 samples (44%). The remainder (hereafter referred to as non-engrafting samples) generated 3 patterns of non-leukemic engraftment: no human graft, defined as

Beschreibung: There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.