Functional genomic landscape of acute myeloid leukaemia.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.

Genomic landscape of neutrophilic leukemias of ambiguous diagnosis.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism.

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70-80% of patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C-terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium-binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL-3-independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2-V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll-like receptor agonist. These effects are not dependent on the novel C-terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis.

ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3ß implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

FANCL ubiquitinates ß-catenin and enhances its nuclear function.

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of ß-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates ß-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, ß-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate ß-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer ß-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/ß-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Glycogen synthase kinase 3beta missplicing contributes to leukemia stem cell generation.

Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.

Integrative analysis of drug response and clinical outcome in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.

Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts.

BACKGROUND: The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. METHODS: Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. RESULTS: Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. CONCLUSION(S): The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic mice. Our data also indicate that expression of either activated Rac1 or Cdc42 alone is not sufficient for malignant transformation of human fibroblasts, although each is required for specific transformed phenotypes. Furthermore, our study elucidates that the expression of several highly significant cancer related genes require the activities of Rac1 and/or Cdc42 which may also play a critical role in cellular transformation.

Efficacy of Ruxolitinib in Patients With Chronic Neutrophilic Leukemia and Atypical Chronic Myeloid Leukemia.

PURPOSE: Colony-stimulating factor-3 receptor (CSF3R)-T618I is a recurrent activating mutation in chronic neutrophilic leukemia (CNL) and to a lesser extent in atypical chronic myeloid leukemia (aCML) resulting in constitutive JAK-STAT signaling. We sought to evaluate safety and efficacy of the JAK1/2 inhibitor ruxolitinib in patients with CNL and aCML, irrespective of CSF3R mutation status. METHODS: We conducted a phase II study of ruxolitinib in 44 patients (21 CNL and 23 aCML). The primary end point was overall hematologic response rate (ORR) by the end of 6 continuous 28-day cycles for the first 25 patients enrolled. We considered a response as either partial (PR) or complete response (CR). We expanded accrual to 44 patients to increase our ability to evaluate secondary end points, including grade ≥ 3 adverse events, spleen volume, symptom assessment, genetic correlates of response, and 2-year survival. RESULTS: ORR was 32% for the first 25 enrolled patients (8 PR [7 CNL and 1 aCML]). In the larger cohort of 44 patients, 35% had a response (11 PR [9 CNL and 2 aCML] and 4 CR [CNL]), and 50% had oncogenic CSF3R mutations. The mean absolute allele burden reduction of CSF3R-T618I after 6 cycles was greatest in the CR group, compared with the PR and no response groups. The most common cause of death is due to disease progression. Grade ≥ 3 anemia and thrombocytopenia were observed in 34% and 14% of patients, respectively. No serious adverse events attributed to ruxolitinib were observed. CONCLUSION: Ruxolitinib was well tolerated and demonstrated an estimated response rate of 32%. Patients with a diagnosis of CNL and/or harboring CSF3R-T618I were most likely to respond.

Two myeloid leukemia cases with rare FLT3 fusions.

Genetic rearrangements involving FLT3 are rare and only recently have been detected in myeloid/lymphoid neoplasms associated with eosinophilia (MLN-eos) and chronic myeloproliferative disorders. Here we report two cases with FLT3 fusions in patients demonstrating mixed features of myelodysplastic/myeloproliferative neoplasms. In the first case, FLT3 was fused with a new fusion partner MYO18A in a patient with marrow features most consistent with atypical chronic myeloid leukemia; the second case involving ETV6-FLT3 fusion was observed in a case with bone marrow features most consistent with chronic myelomonocytic leukemia. Notably, we observed that samples from both patients demonstrated FLT3 inhibitor (quizartinib and sorafenib) sensitivity in ex vivo drug screening assay.

Myelodysplastic Syndromes: Updates and Nuances.

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal stem cell disorder of the blood and marrow typically diagnosed based on the presence of persistent cytopenia(s), dysplastic cells, and genetic markers. Common issues that arise in the clinical management include difficulty confirming MDS diagnosis, lack of a standard approach with novel agents in MDS, and few prospective long-term, randomized controlled MDS clinical studies to guide allogeneic blood and marrow transplant. With the recent genetic characterization of MDS, certain aspects of these issues will be better addressed by integrating genetic data into clinical study design and clinical practice.

Recent Progress in Chronic Neutrophilic Leukemia and Atypical Chronic Myeloid Leukemia.

PURPOSE OF REVIEW: We reviewed recent diagnostic and therapeutic progress in chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML). We summarized recent genetic data that may guide future efforts towards implementing risk-adapted therapy based on mutational profile and improving disease control and survival of affected patients. RECENT FINDINGS: Recent genetic data in CNL and aCML prompted modifications to the World Health Organization (WHO) diagnostic criteria, which have improved our understanding of how CNL and aCML are different diseases despite sharing common findings of peripheral granulocytosis and marrow myeloid hyperplasia. The overlap of recurrently mutated genes between aCML and CMML support considering CSF3R-T618I mutated cases as a distinct entity, either as CNL or CNL with dysplasia. Ongoing preclinical and clinical studies will help to further inform the therapeutic approach to these diseases. Our understanding of CNL and aCML has greatly advanced over the last few years. This will improve clarity for the diagnosis of these diseases, provide a strategy for risk stratification, and guide risk-adapted therapy.

What's different about atypical CML and chronic neutrophilic leukemia?

Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) are rare myeloid neoplasms defined largely by morphologic criteria. The discovery of CSF3R mutations in aCML and CNL have prompted a more comprehensive genetic profiling of these disorders. These studies have revealed aCML to be a genetically more heterogeneous disease than CNL, however, several groups have reported that SETBP1 and ASXL1 mutations occur at a high frequency and carry prognostic value in both diseases. We also report a novel finding-our study reveals a high frequency of U2AF1 mutations at codon Q157 associated with CSF3R mutant myeloid neoplasms. Collectively, these findings will refine the WHO diagnostic criteria of aCML and CNL and help us understand the genetic lesions and dysregulated signaling pathways contributing to disease development. Novel therapies that emerge from these genetic findings will need to be investigated in the setting of a clinical trial to determine the safety and efficacy of targeting various oncogenic drivers, such as JAK1/2 inhibition in CSF3R-T618I-positive aCML and CNL. In summary, recent advances in the genetic characterization of CNL and aCML are instrumental toward the development of new lines of therapy for these rare leukemias that lack an established standard of care and are historically associated with a poor prognosis.

Durable Disease Control with MEK Inhibition in a Patient with NRAS-mutated Atypical Chronic Myeloid Leukemia.

Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) are rare hematologic neoplasms characterized by leukocytosis and a hypercellular bone marrow. Although recurrent mutations in the colony-stimulating factor 3 receptor (CSF3R) are frequently observed in patients with (CNL), the mutational landscape in (aCML) is less well-defined. In this report, we describe an 81-year-old male who was diagnosed with aCML. He presented with leukocytosis and anemia but no significant clinical symptoms. Standard laboratory studies revealed the absence of the Philadelphia chromosome. Massively parallel sequencing demonstrated no mutations in CSF3R, but the presence of a heterozygous NRAS-G12D variant (47% allele frequency). The patient was started on treatment with trametinib, an MEK1/2 inhibitor with Food and Drug Administration approval for malignant melanoma. Therapy with trametinib resulted in exceptional improvements in his blood counts and continued disease control with 14 months of follow-up. This case highlights the need for clinical trials evaluating the safety and efficacy of MEK1/2 as a therapeutic target for the treatment of patients with NRAS-mutated aCML/CNL.

Significant clinical response to JAK1/2 inhibition in a patient with CSF3R-T618I-positive atypical chronic myeloid leukemia.

Mutations in CSF3R (colony-stimulating factor 3 receptor) are frequent oncogenic drivers in chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML). Here we describe a 75 year old man who was diagnosed with CSF3R-T618I-positive atypical CML. He presented with leukocytosis, anemia, and thrombocytopenia and developed massive splenomegaly and severe constitutional symptoms. Hydroxyurea was given over a 6 month period but failed to provide any measureable clinical benefit. Eventually, he was treated with ruxolitinib, an FDA-approved JAK1/2 inhibitor, which resulted in dramatic improvement of his blood counts. He also had significant reduction of spleen volume and constitutional symptoms. This case highlights the need for a clinical trial to interrogate JAK1/2 as a potential molecular target in CNL and aCML in patients with or without CSF3R mutation. A clinical trial evaluating the safety and efficacy of ruxolitinib for this patient population is registered at ClinicalTrials.gov (NCT02092324).

Next-generation medicine: combining BCR-ABL and Hedgehog-targeted therapies.

Disease relapse remains a major cause of death in patients with BCR-ABL-positive leukemia despite advances in treatment with kinase inhibitors. Significant efforts are underway to target pathways that maintain leukemia stem cells. Targeting these pathways holds promise for definitive leukemia eradication or improvement of the effectiveness of currently available therapies.

The PI3K/Akt1 pathway enhances steady-state levels of FANCL.

Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48-linked chains. Evaluation of a series of N-terminal-deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3ß is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3ß. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function.