Heterogeneity Of Acute Myeloid Leukemia Research Articles

MLN0128, a Second-Generation mTOR Kinase Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML

Abnormal mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active site mTOR kinase inhibitors (TOR-KI) represent a promising new approach to target the PI3K/AKT/mTOR pathway. In a previous study, we used a synthetic TOR-KI PP242 to demonstrate that disrupting AKT/mTOR signaling with this class of compounds can effectively target leukemic cells under conditions mimicking bone marrow microenvironment[1]. In the current study, we focused on the inhibitory mechanisms in rare and heterogeneous AML stem cells and on the cellular antagonistic responses after mTORC1/2 blockage using MLN0128, a synthetic TOR-KI currently in clinical development. We found that MLN0128 inhibited cell proliferation and induced apoptosis in AML cells by attenuating mTOR complex 1 and 2 (mTORC1/2) activities, and showed higher anti-leukemia potency than PP242. Using high-throughput time-of-flight mass cytometry (CyTOF)[2], combined with advanced computational analytic software Spanning-Tree Progression of Density-Normalized Events (SPADE)[3] and visualization t-Distribution Stochastic Neighbor Embedding (viSNE)[4], we identified AML subsets co-expressing multiple stem cell markers with high AKT/mTOR activity. MLN0128 selectively targets mTORC1/2 downstream signaling in these cells with minimal effects on other pathways, and lacks the effect on non- leukemic stem cells (LSC). The inhibitory effect of MLN0128 on LSC is positively correlated to that of a number of co-expressing stem cell molecules, and to the baseline expression of and the degree of SCF activation on AKT/mTOR signaling. Using the reverse phase protein array (RPPA) technique, we studied expression and phosphorylation changes in 151 proteins from 24 primary AML samples in response to MLN0128, and revealed several pro-survival pathways may protect from MLN0128-induced cellular stress. We identified four druggable targets that were significantly triggered by MLN0128 inhibition: PARP (that can be targeted by PARP inhibitor olaparib), HDAC3 (vorinostat), p-SRC (dasatinib), and STAT5 (ruxolitinib). Blocking AKT/mTOR signaling and these pro-survival cascades triggered complex responses in AML cell lines and primary AML samples, resulting in an overall increase in apoptosis of AML cells compared to single target inhibition. Among the four combinations, MLN0128 and vorinostat elicited the best responses. In summary, our findings indicate that blocking AKT/mTOR signaling with the mTORC1/2 inhibitor MLN0128 causes growth inhibition and apoptosis in AML cells. High-throughput CyTOF technology revealed that MLN0128 selectively targets primary AML stem cells with high AKT/mTOR activity. RPPA mapping multiple intracellular signaling pathways in primary AML cells in response to mTORC1/2 inhibition allowed us to identify compensatory activation of actionable pro-survival pathways. Combinatorial multi-targeted approaches demonstrated various responses to MLN0128 in AML samples, suggesting that personalized treatment selections will be needed in individual AML cases. Finally, utilization of techniques such as CyTOF or RPPA may assist in profiling intra-cellular and inter-patient variability of responses to targeted agents which likely stem from multiple genetic and epigenetic events within individual tumor, and serve as tools for selection of individualized targeted therapies in AML and other malignancies.Reference1. Zeng, Z.H., et al., Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment. Blood, 2012. 120(13): p. 2679-2689.2. Bendall, S.C., et al., Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science, 2011. 332(6030): p. 687-696.3. Qiu, P., et al., Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nature Biotechnology, 2011. 29(10): p. 886-U181.4. Amir el, A.D., et al., viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol, 2013. 31(6): p. 545-52. DisclosuresNo relevant conflicts of interest to declare.

Intermediate-risk acute myeloid leukemia therapy: current and future

Abstract In recent years, research in molecular genetics has been instrumental in deciphering the molecular heterogeneity of acute myeloid leukemia (AML), in particular the subset of patients with “intermediate-risk” cytogenetics. However, at present, only the markers NPM1, CEBPA, and FLT3 have entered clinical practice. Treatment of intermediate-risk AML patients eligible for intensive therapy has not changed substantially. The “3 + 7” induction therapy still represents the standard of care. The addition of the immunoconjugate gemtuzumab ozogamicin to therapy has been shown to improve outcome; however, the drug is not approved for this use. A common standard for postremission therapy is the administration of repeated cycles of intermediate- to high-dose cytarabine. Allogeneic stem cell transplantation may offer a survival benefit for many patients with intermediate-risk AML. Patients are best selected based on the genetic profile of the leukemia cells and the risk associated with the transplantation itself. A myriad of novel agents targeting mutant leukemia drivers or deregulated pathways are in clinical development. In the past, many novel compounds have not met expectations; nonetheless, with the rapid developments in comprehensive molecular profiling and new drug design, there is the prospect of personalizing therapy and improving patient outcome.

Intermediate-risk acute myeloid leukemia therapy: current and future.

In recent years, research in molecular genetics has been instrumental in deciphering the molecular heterogeneity of acute myeloid leukemia (AML), in particular the subset of patients with "intermediate-risk" cytogenetics. However, at present, only the markers NPM1, CEBPA, and FLT3 have entered clinical practice. Treatment of intermediate-risk AML patients eligible for intensive therapy has not changed substantially. The "3 + 7" induction therapy still represents the standard of care. The addition of the immunoconjugate gemtuzumab ozogamicin to therapy has been shown to improve outcome; however, the drug is not approved for this use. A common standard for postremission therapy is the administration of repeated cycles of intermediate- to high-dose cytarabine. Allogeneic stem cell transplantation may offer a survival benefit for many patients with intermediate-risk AML. Patients are best selected based on the genetic profile of the leukemia cells and the risk associated with the transplantation itself. A myriad of novel agents targeting mutant leukemia drivers or deregulated pathways are in clinical development. In the past, many novel compounds have not met expectations; nonetheless, with the rapid developments in comprehensive molecular profiling and new drug design, there is the prospect of personalizing therapy and improving patient outcome.

Treating acute myeloid leukemia in older adults.

Acute myeloid leukemia (AML) is a disease of older adults, with a median age of 67 years at presentation. In the past, only a third of older patients (defined as individuals older than 60-65 years of age) with AML received definitive therapy for their disease due to concerns about their overall fitness and potential treatment-related mortality. However, compelling epidemiological data have shown unequivocally that older AML patients up to 80 years old both tolerate and survive longer after therapy than their untreated counterparts. Current therapeutic options for elderly individuals with AML include intensive chemotherapy with a cytarabine and anthracycline backbone, hypomethylating agents (decitabine and azacitidine), low-dose cytarabine, investigational agents, and supportive care with hydroxyurea and transfusions. Over the last few years, there has been increasing debate regarding the appropriate therapeutic approach to take in older adults given the diversity of the geriatric patient population and heterogeneous AML disease biology. This article discusses how performance status, comorbidities, disease characteristics, quality of life concerns, and long-term treatment goals affect the selection of appropriate therapy for older adults with AML. Risks and benefits of each treatment approach based on the most recent medical literature are discussed. Finally, a treatment algorithm summarizing these data and incorporating geriatric assessment and molecular and cytogenetic markers predictive of therapeutic response is proposed to aid in the clinical decision-making process.

Combined Molecular and Clinical Prognostic Index for Relapse and Survival in Cytogenetically Normal Acute Myeloid Leukemia

Cytogenetically normal (CN) acute myeloid leukemia (AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician, it is difficult to summarize the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pretreatment patient and disease characteristics. Two prognostic indices for CN-AML (PINA), one regarding overall survival (OS; PINAOS) and the other regarding relapse-free survival (RFS; PINARFS), were derived from data of 572 patients with CN-AML treated within the AML Cooperative Group 99 study (www.aml-score.org). On the basis of age (median, 60 years; range, 17 to 85 years), performance status, WBC count, and mutation status of NPM1, CEBPA, and FLT3-internal tandem duplication, patients were classified into the following three risk groups according to PINAOS and PINARFS: 29% of all patients and 32% of 381 responding patients had low-risk disease (5-year OS, 74%; 5-year RFS, 55%); 56% of all patients and 39% of responding patients had intermediate-risk disease (5-year OS, 28%; 5-year RFS, 27%), and 15% of all patients and 29% of responding patients had high-risk disease (5-year OS, 3%; 5-year RFS, 5%), respectively. PINAOS and PINARFS stratified outcome within European LeukemiaNet genetic groups. Both indices were confirmed on independent data from Cancer and Leukemia Group B/Alliance trials. We have developed and validated, to our knowledge, the first prognostic indices specifically designed for adult patients of all ages with CN-AML that combine well-established molecular and clinical variables and that are easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care through risk-adapted therapy of CN-AML.

Functional Heterogeneity of Genetically Defined Subclones in Acute Myeloid Leukemia

The relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by whole-genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.

The AML Salad Bowl

Tumors arise from single cells but become genetically heterogeneous through continuous acquisition of somatic mutations as they progress. In this issue of Cancer Cell, Klco and colleagues used whole genome sequence analysis to demonstrate the correlation of genetic clonal architecture with functional heterogeneity in acute myeloid leukemia.

Single-Cell Mass Cytometry Reveals Phenotypic and Functional Heterogeneity In Acute Myeloid Leukemia At Diagnosis and In Remission

Acute myeloid leukemia (AML) is organized in a hierarchy with a rare population known as leukemia stem cells (LSC) capable of self-renewal and propagation of the disease. Characterization of the unique phenotypes and complex signaling pathways in LSCs that survive induction chemotherapy is essential for understanding of the mechanisms of chemoresistance and designing the strategies to eliminate residual leukemia clones.In this study, we compared signaling profiles of distinct phenotypic AML subsets in paired bone marrow (BM) samples collected at diagnosis and after achieving the complete remission (CR). Cell surface characteristics and signaling pathways activated within sub-populations of AML samples were defined using the novel technology of time-of-flight mass cytometry (CyTOF) that has the ability to perform up to 100 mutiparameter assays in single cells (Bendall et al, Science 2011). First, we validated CyTOF measurements by performing cross-comparisons of surface markers and intracellular proteins measured in AML cells with traditional multi-parametric flow cytometry (FCM). Frequencies of CD123+CD99+ population within CD34+CD38- cells were 73.7%±1.8% and 78.5%±3.7% by CyTOF and FCM. Patterns of specific activation of the intracellular proteins pSTAT5, pERK1/2 and pAKT by GM-CSF, PMA and SCF, and inhibition by selective kinase inhibitors showed excellent cross-platform consistency between CyTOF, FCM and immunoblotting. Next, mononuclear cells of 5 paired AML (at diagnosis and in CR) and of 3 normal BM (NBM) were stained with 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). A SPADE (spanning-tree progression analysis of density-normalized events) (Qiu et al, Nat Biotechnol. 2011) tree plot was generated, representing clustered expression of the cell-surface antigens. Boundaries and annotations of the AML cells were manually defined to represent distinct cell subsets (Figure 1). We used the pooled data from NBM samples, which showed identical patterns, as a reference. SPADE analysis revealed several subsets unique to the diagnostic AML samples, which were eliminated by chemotherapy; and phenotypically distinct subsets in diagnostic samples that persisted in CR. Notably, a subset defined by the “traditional LSC” markers (CD45dimCD34+CD38lowCD90-CD33-CD117+; annotation #2) was readily identified in diagnostic samples and was significantly reduced by induction chemotherapy in 2 of the 5 AML samples. In one of these samples we identified a distinct subset co-expressing LSC markers CD45dimCD34+CD38lowCD33-CD117-CD99lowCD133low (annotation #3) that was present in both diagnostic (1.1%) and CR (1.7%) BM; this subset may have contributed to the MRD detected by standard leukemia-associated immunophenotypes. [Display omitted] We next investigated intracellular signaling pathways in antigen-defined AML subpopulations using CyTOF. Activation of p-AKT and pS6 showed similar pattern in subsets defined by annotations 1, 9 and 10 at diagnosis (Figure 2A), and was largely reduced in the CR BM. In turn, activation of p-4EBP1 and p-mTOR were observed in multiple subsets (#1-5 and 9-11) in all diagnostic AML samples, especially in a subset 1 characterized by the “Progenitor” phenotype, and remained heightened in the CR samples (Figure 2B). [Display omitted] In summary, using CyTOF and SPADE, we characterized phenotype-specific intracellular signaling pathways in AML samples at diagnosis and in CR. Persistent activation of p-mTOR and p-4EBP1 are identified in the subpopulations of AML progenitors in CR, and may present the potentially targetable pathways in AML. The study is ongoing with prospective CyTOF analysis of a larger set of paired AML samples at diagnosis, CR and relapse coupled with the molecular analysis of the distinct subpopulations. Disclosures:No relevant conflicts of interest to declare.

Proof-Of-Concept Study For Precision Medicine With Chromosome Genomic Array Testing (CGAT) For Drug Sensitivity Screening In Acute Myeloid Leukemia

Tremendous heterogeneity in acute myeloid leukemia (AML) poses a significant challenge to clinical management and effective therapy. Cytogenetics is one of the key prognostic factors for AML. New technologies, such as chromosome genomic array testing (CGAT) combining both SNP-based and non-SNP based array CGH, have allowed informative genomic profiling of chromosome abnormalities with very high resolution. Yet it remains unclear how these unique CGAT-identified clonal aberrations impact the outcome of AML patients and how they predict a patient's response to a specific medication. In this proof-of-concept study, we aim to explore the correlation of genomic abnormalities identified by CGAT with in vitrodrug sensitivity data from 30 AML patients and their clinical responses.We performed CGAT on the pre-treatment bone marrow of these AML patients using CytoScanHD, which includes 2.5 million markers, 750K of which are SNP markers. Copy number aberrations (CNA) and copy-neutral loss of heterozygosity (cnLOH) were analyzed by ChAS and Nexus software. CGAT abnormalities were enumerated based on chromosome arms affected; i.e., multiple segmental CNAs and cnLOH on the same chromosome arm were counted as one aberration. Patients' response to chemotherapy and clinical relapse were tracked. Drug sensitivity for individual patient leukemia blast samples was measured by a high-throughput in vitroassay consisting of a panel of 160 drugs, of which 45 are FDA approved and 115 investigational. Cell survival was measured by Cell Titer Glo, and data curves for 8 concentrations of each drug were generated to derive EC50 and IC50 values. Statistical correlation was analyzed by Fisher's exact test, Cox regression, and regression analysis.CGAT detected all the chromosome abnormalities identified by cytogenetics except truly balanced translocations. CGAT also identified clonal aberrations in 7 of the 13 patients with normal cytogenetics. Of the 6 uniquely CGAT abnormal patients, 3 demonstrated cnLOH only and 2 CNA only. Overall, CGAT showed 11 complex karyotypes (CK, defined as >=3 aberrations; high-risk) and 19 normal or simple abnormal cases (low/intermediate-risk), as compared to the cytogenetics classification of 9 poor-risk, 15 intermediate-risk, and 6 low-risk karyotypes. cnLOH was detected in 9 patients (30%). Complete remission (CR) rate after the first induction course in the CGAT CK group was 18% versus 68% in the non-CK group (P=0.02). There was a very high concordance (90%) between CGAT risk based on CK and conventional cytogenetics. And when cytogenetics and CGAT disagree, as for the 2 patients who had normal cytogenetics but CGAT CK, CGAT risk stratification appeared better as neither patient achieved CR. CR duration longer than 1 year occurred more (P=0.04) in the CGAT normal patients (50%) than in the CGAT abnormal patients (8.3%). Among those who achieved CR, the hazard of relapse (HR) for CGAT CK patients was 9.9 (CI: 1.6-61; P=0.02); HR for cnLOH patients was 7.1 (CI: 1.2-43; P=0.03). Considering the total aberrations (median 1.5, range 0-16), the HR for relapse among CR patients was 1.3 (CI: 1.1-1.5; P=0.01) per aberration. Six of the 48 drugs that exhibited cytotoxicity in the in vitro assay, including clofarabine (r=0.45, P=0.01) and PKI-587 (dual PI3K/mTOR inhibitor) (r=0.48, P=0.008), showed significant correlation between CGAT aberrations and EC50; i.e, the more CGAT aberrations, the higher concentration is required for a given drug to kill these CGAT-abnormal leukemic cells in vitro.In conclusion, CGAT increased the diagnostic yield by 54% among AML patients with normal cytogenetics and altered the overall risk classification by 6.7%. Complex karyotype identified by CGAT carried the same poor prognostic effect as CK by cytogenetics. Although abnormal CGAT results did not appear to predict response, increasing CGAT abnormalities are associated with early relapse, as is the presence of cnLOH. Genomic complexity detected by CGAT appeared to associate with dosage-dependent resistance to selected drugs by in vitro drug sensitivity analyses. Characterization of prognostic effect and drug response associated with each type of CGAT abnormalities will require a larger data set. Nevertheless, this study demonstrated the clinical utility of CGAT for precision medicine in AML. Disclosures:Becker:Affymetrix: Research Funding.

The Applicability Of The WHO Classification In Pediatric AML. A NOPHO-AML Study

IntroductionThe World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia was revised in 2008 and is based on genetic characteristics and morphology. The classification incorporates newly recognized entities and emphasizes, more than previously, the pivotal role of cytogenetic abnormalities aiming at identifying biological and clinical entities as basis for a stratified treatment. The classification of acute myeloid leukemia (AML) is primarily based on adult studies. However, the frequency of cytogenetic changes varies among children and adults and the relevance of the 2008 revised WHO classification in pediatric AML has not yet been elucidated. The aim of this study was to evaluate the usability of the WHO classification when applied to a large population-based pediatric AML cohort. MethodsWe included children 0-18 years of age diagnosed with de novo AML in the Nordic countries (Sweden, Norway, Finland, Iceland and Denmark) and Hong Kong treated according to the NOPHO-AML-1993 and 2004 protocols in the period January 1993 to December 2012. Clinical, morphologic, and genetic data were retrieved from the database. Patients with myeloid leukemia of Down syndrome, acute promyelocytic leukemia, and therapy-related AML were excluded. The karyotypes from the Nordic countries were centrally reviewed with annual evaluation of the karyograms by the NOPHO cytogenetic working group. Morphological and molecular analyses and immunophenotyping were carried out at regional centers. The patients were classified according to the revised 2008 WHO classification using genetic parameters and the French-American-British (FAB) classification. ResultsIn the NOPHO database we identified 609 children aged 0-18 years diagnosed with AML. In 13 cases, the karyotype analyses either failed or considered uninformative and were excluded. Among the remaining 596 informative cases (98%), 241 (40%) were categorized as AML with recurrent genetic abnormalities; t(8;21)(q22;q22);RUNX1-RUNX1T1 (n=69), inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 (n=47), t(9;11)(p21;q23);MLLT3-MLL (n=61), t(6;9)(p22;q34);DEK-NUP214 (n=5), inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI1 (n=1), t(1;22)(p13;q13)/RBM15-MKL1 (n=4), NPM1 mutated (n=13), and CEBPA mutated (n=10). Furthermore, 23 children were classified in the provisional entity AML with FLT3-ITD. Based on the karyotype, 92 children (16%) were classified as AML with myelodysplasia-related cytogenetical changes (AML-MDS). This group was dominated by unbalanced abnormalities (9%) with -7/del(7q) being the most common (4% of the total cohort) and complex karyotypes (6% of the total cohort). AML with balanced myelodysplasia-related changes was found in 0.5%. The largest group was the highly heterogeneous AML not otherwise specified (AML-NOS) (n=263) (44%), and included some of the well-defined 11q23 abnormalities. In the 2001 version of the WHO classification, “AML with abnormalities of 11q23; MLL,” was listed separately, but in the 2008 edition only AML with t(9;11)(p22;q23);MLLT3-MLL was listed as an entity. The AML-NOS cases were sub-classified according to morphology, with FAB M5 being most common, found in 22% of NOS, followed by FAB M1, FAB M2, and FAB M4, all found in 18%.In conclusion, 40% of the children were classified in clinically relevant entities, but the WHO classification allocated 16% of the children to AML-MDS, the relevance is unknown in children, and 44% to the NOS group. This large and unspecified group limits the applicability of the WHO classification in children with AML suggesting that additional considerations are warranted for relevant classification of pediatric AML. Disclosures:No relevant conflicts of interest to declare.

The prognostic relevance of miR-212 expression with survival in cytogenetically and molecularly heterogeneous AML

Acute myeloid leukemia (AML) is a highly heterogeneous disease, characterized by various cytogenetic and molecular abnormalities, many of which may express prognostic value. MicroRNAs (miRNAs) are a class of small regulatory RNAs. The prognostic value of miRNAs in AML is yet to be determined. Here, we set out to identify miRNAs that are consistent significant prognostic determinants, independent from other known prognostic factors. A discovery cohort (n=167) and validation cohort (n=409) of a heterogeneous AML population were used to reliably identify miRNAs with prognostic value. We report miR-212 as an independent prognostic factor, significantly associated with a prolonged overall survival (OS) and also event-free and relapse-free survival in a discovery cohort (hazard ratio (HR)s=0.77, P=0.015 for OS) that was subsequently confirmed in an independent validation cohort of 409 cases (HR=0.83, P=0.016). The prognostic significance and the prevalence of high miR-212 did not correlate with specific (cyto)genetic subtypes of AML. High miR-212 expression levels are associated with a gene expression profile that is significantly enriched for genes involved in the immune response. MiR-212 may improve the current prognostic risk stratification of mixed AML including normal karyotype AML and AML with cytogenetic and molecular abnormalities.

A Noncanonical Flt3ITD/NF-κB Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia

Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML.

Microdeletions at Apparently Balanced Translocation Breakpoints Are Biomarkers for Heightened Genomic Instability in Patients with Non-MLL Acute Myeloid Leukemia

Abstract 2529The clinical heterogeneity of acute myeloid leukemia (AML) is well established and identification of specific balanced translocations is a critical component of the classification of AML. Patients with translocations are assigned to prognostic groups generally defined as good [e.g., t(8;21)(q22;q22), inv(16) (p13q22), t(15;17)(q24;q21)], intermediate [e.g., t(3;5)(q25;q34), t(9;11)(p22;q23)] and poor [e.g., other MLL translocations, inv(3)(q21q26.2), t(6;9)(p23;q34)]. Further refinement based on the identification of new genetic markers may help tailor prognostic and treatment decisions. In an effort to identify new molecular markers associated with recurrent translocations, we have analyzed 30 cases of AML with apparently balanced translocations using a microarray-based technology coupled with linear DNA amplification [translocation-CGH (tCGH)], providing high resolution mapping of translocation breakpoints and identification of copy number abnormalities at those breakpoints (BkPt-CNAs). Analysis can be multiplexed to interrogate 14 balanced translocations in a single test. Additionally, all cases were assayed by high-density aCGH to identify additional clinically relevant copy number abnormalities (CNAs) throughout the genome. For each sample these test results were compared. Patients were all adults (>40 years of age), 10 males and 20 females, and included t(15;17)(q24;q21) (8 cases), t(8;21) (q22;q22) (6 cases), inv(16)(p13q22) (5 cases), t(9;11)(p22;q23) (5 cases), t(3;5)(q25;q34) (4 cases), t(6;11)(q27;q23) (1 case) and t(6;9)(p23;q34) (1 case).Of the 30 cases, 6 showed BkPt-CNAs in one or both translocation partners. All 6 (100%) of these cases exhibited additional clinically relevant CNAs, whereas only 11/24 (46%) cases with no BkPt-CNAs showed additional CNAs. Thus, BkPt-CNAs are significantly correlated with the presence of secondary clinically relevant alterations (p=0.0208). Importantly, no MLL translocations cases revealed BkPt-CNAs. In contrast, 3 of 4 t(3;5)(q25;q34) cases showed BkPt-CNAs in both genes (NPM1 and MLF1) and additional CNAs, with two cases showing large deletions (>0.5 Mb) at both breakpoints. Alterations involved RSRC1 (mRNA splicing), FBXW11 (E3 ubiquitin protein ligase), STK10 (serine/threonine kinase), FGFR3 (growth factor receptor) and MGMT (O-6-methylguanine-DNA methyltransferase), findings potentially relevant to disease progression and treatment response. Cases of t(15;17), t(8;21) and inv(16) each showed a single case with BkPt-CNAs and these correlated with greater complexity for additional CNAs. For t(15;17), that case showed partial duplication of both the PML and RARA genes (47 kb and 39 kb) yielding a cryptic fusion (missed by karyotype and FISH) that produced the “long” form (bcr1) transcript based on the PML breakpoint and confirmed by rtPCR. This case also had a 24.56 Mb deletion at 13q14.2->q21.33 involving RB1, MIR15A and MIR16-1. In the 7 remaining PML-RARA cases [4 short (bcr3) and 3 long (bcr1)] with no BkPt-CNAs, two cases showed non-complex CNAs [(e.g., -Y (bcr1) or +8 (bcr3)]. Five of six t(8;21) cases exhibited CNAs, but the case with a BkPt-CNA (39 kb within RUNX1) exhibited greater complexity for CNAs (multiple gains and losses over >70 MB of distal 9q) than other cases (e.g., +8 or +15). Similarly, analysis of inv(16) cases demonstrated CNAs in 3 of 5 cases, with the greatest complexity in the case with BkPt-CNAs [deletion in CBFB and MYH11 involving ABCC1 (anion transporter implicated in multiple drug resistance), deletions 9q34.13 involving ABL1 and NUP214], while other cases had single changes of a homozygous deletion of GSTT1 or a heterozygous deletion of the 16p11.2 microdeletion syndrome region, either potentially inherited. The sole case of t(6;9) had only the relevant translocation.In conclusion, analysis of AML cases with balanced translocation by t-CGH and aCGH revealed significant correlation between BkPt-CNAs and the presence of secondary CNAs. Moreover, when BkPt-CNAs were present, the molecular complexity of additional CNAs was increased. This observation held true across various translocations evaluated, with the notable exception of MLL translocations. Thus BkPt-CNAs provide a broadly applicable signature for a subtle molecular instability in AML that, acquired or inherited, may predate translocations and may prove relevant to treatment response and outcome. Disclosures:Schultz:Signature Genomic Laboratories: Employment. Campbell:Abbott Molecular: Honoraria. Furrow:Signature Genomic Laboratories: Employment. Shaffer:Signature Genomic Laboratories: Employment. Ballif:Signature Genomic Laboratories: Employment.

Activity of the multikinase inhibitor sorafenib in combination with cytarabine in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous cancer that frequently exhibits aberrant kinase signaling. We investigated a treatment strategy combining sorafenib, a multikinase inhibitor with limited single-agent activity in AML, and cytarabine, a key component of AML chemotherapy. Using 10 human AML cell lines, we determined the effects of sorafenib (10 μM) on antileukemic activity by measuring cell viability, proliferation, ERK1/2 signaling, and apoptosis. We also investigated the effects of sorafenib treatment on the accumulation of cytarabine and phosphorylated metabolites in vitro. A human equivalent dose of sorafenib in nontumor-bearing NOD-SCID-IL2Rγ(null) mice was determined by pharmacokinetic studies using high performance liquid chromatography with tandem mass spectrometric detection, and steady-state concentrations were estimated by the fit of a one-compartment pharmacokinetic model to concentration-time data. The antitumor activity of sorafenib alone (60 mg/kg) twice daily, cytarabine alone (6.25 mg/kg administered intraperitoneally), or sorafenib once or twice daily plus cytarabine was evaluated in NOD-SCID-IL2Rγ(null) mice bearing AML xenografts. Sorafenib at 10 μM inhibited cell viability, proliferation and ERK1/2 signaling, and induced apoptosis in all cell lines studied. Sorafenib also increased the cellular accumulation of cytarabine and metabolites resulting in additive to synergistic antileukemic activity. A dose of 60 mg/kg in mice produced a human equivalent sorafenib steady-state plasma exposure of 10 μM. The more dose-intensive twice-daily sorafenib plus cytarabine (n = 15) statistically significantly prolonged median survival in an AML xenograft model compared with sorafenib once daily plus cytarabine (n = 12), cytarabine alone (n = 26), or controls (n = 27) (sorafenib twice daily plus cytarabine, median survival = 46 days; sorafenib once daily plus cytarabine, median survival = 40 days; cytarabine alone, median survival = 36 days; control, median survival = 19 days; P < .001 for combination twice daily vs all other treatments listed). Sorafenib in combination with cytarabine resulted in strong anti-AML activity in vitro and in vivo. These results warrant clinical evaluation of sorafenib with cytarabine-based regimens in molecularly heterogeneous AML.

Identification of new markers discriminating between myeloid and lymphoid acute leukemia

Background: The heterogeneity of acute myeloid leukemia (AML) with respect to biology and clinical course resides in the fact that patients belonging to the same group show marked differences in their response to chemotherapy, necessitating a refinement of AML classification.Methods: In order to define molecular markers for AML, we performed microarray analysis on peripheral blood cells from two M5 AML patients, and selected four differentially expressed genes to validate their expression by real-time quantitative PCR (RT-PCR).Results: We have shown that two downregulated genes in AML, those encoding guanine nucleotide-binding protein γ11 (GNG11) and amphiregulin (AREG), are also downregulated in B-lineage acute lymphoblastic leukemia (B-ALL) and T-lineage acute lymphoblastic leukemia (T-ALL) patients. A second gene, that encoding ceruloplasmin (CP), is upregulated in AML but not in B-ALL and T-ALL. The level of expression of these genes varies from one patient to another.Conclusion: Since the number of patients studied is limited, further studies are needed with a larger series of patients to evaluate the potential utility of GNG11, AREG and CP as molecular markers for AML subtype classification. Our study is the first to analyze these genes in AML, B-ALL, T-ALL and chronic leukemia (myeloid and lymphoid) patients by RT-PCR. This rapid and sensitive method could be used to screen these genes in different types of leukemia.

Dasatinib Inhibits the Growth of Molecularly Heterogeneous Myeloid Leukemias

Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x 10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Genes of cell-cell interactions, chemotherapy detoxification and apoptosis are induced during chemotherapy of acute myeloid leukemia

BackgroundThe molecular changes in vivo in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response.MethodsThe gene expression was examined by whole genome 44 k oligo microarrays and 12 k cDNA microarrays in peripheral blood leukocytes collected from seven leukemia patients before treatment, 2–4 h and 18–24 h after start of chemotherapy and validated by real-time quantitative PCR. Statistically significantly upregulated genes were classified using gene ontology (GO) terms. Parallel samples were examined by flow cytometry for apoptosis by annexin V-binding and the expression of selected proteins were confirmed by immunoblotting.ResultsSignificant differential modulation of 151 genes were found at 4 h after start of induction therapy with cytarabine and anthracycline, including significant overexpression of 31 genes associated with p53 regulation. Within 4 h of chemotherapy the BCL2/BAX and BCL2/PUMA ratio were attenuated in proapoptotic direction. FLT3 mutations indicated that non-responders (5/7 patients, 8 versus 49 months survival) are characterized by a unique gene response profile before and at 4 h. At 18–24 h after chemotherapy, the gene expression of p53 target genes was attenuated, while genes involved in chemoresistance, cytarabine detoxification, chemokine networks and T cell receptor were prominent. No signs of apoptosis were observed in the collected cells, suggesting the treated patients as a physiological source of pre-apoptotic cells.ConclusionPre-apoptotic gene expression can be monitored within hours after start of chemotherapy in patients with acute myeloid leukemia, and may be useful in future determination of therapy responders. The low number of patients and the heterogeneity of acute myeloid leukemia limited the identification of gene expression predictive of therapy response. Therapy-induced gene expression reflects the complex biological processes involved in clinical cancer cell eradication and should be explored for future enhancement of therapy.

Heterogeneity of Acute Myeloid Leukemia at the Stem Cell Level.

Leukemias are considered hierarchically organized being maintained by a leukemia stem cell (LSC). Whereas LSCs become the primary focus for targeted therapies, little is known about the pathways regulating LSC fidelity. Using retroviral gene transfer of MN1, NUP98HOXD13 (ND13), or HOXA9 oncogenes and limiting-dilution transplantation we modelled leukemias with different LSC frequencies, and characterized critical signaling pathways by loss-of-function analysis. Here we establish the concept that LSCs are heterogeneous based on the number of activated transcriptional networks, and functionally characterize downstream targets that are critical for LSC activity. Constitutive expression of the very potent myeloid oncogene MN1 with the ND13 fusion gene in murine bone marrow cells results in acute myeloid leukemia (AML) that is phenotypically very similar to MN1-induced AML. However, limiting dilution analysis showed that the LSC frequency was 33 fold higher in MN1+ND13 cells compared to MN1 cells, and disease latency at the limiting dilution was significantly shorter in the combination model (p=.009). Whereas MN1-LSCs expanded 68-fold over a period of 6 days, MN1+ND13-LSCs expanded 131-fold more than MN1-LSCs as determined by the competitive repopulation unit (CRU) assay. To screen for functional differences of the two models we screened for differential cytokine responses in vitro. Interestingly, MN1+ND13 expressing cells proliferated in response to GM-CSF, whereas MN1 cells or ND13 cells did not. This was confirmed as well for MN1+HOXA9 expressing cells and their MN1+CTL or HOXA9+CTL expressing counterparts. We found that Stat1, Stat3, Stat5, and Erk1/2 were selectively phosphorylated upon cytokine stimulation in MN1+ND13 and MN1+HOXA9 cells compared to single-oncogene transduced cells. To test the role of Stat1 and Stat5b for LSC fidelity Stat1 −/− and Stat5b −/− cells were co-transduced with MN1 and HOXA9 and compared to wildtype cells in vitro and in vivo. Stat1 −/− cells transduced with MN1+HOXA9 proliferated slower than wildtype cells in response to GM-CSF but not with IL3/IL6/SCF. Proliferation of Stat5b −/− cells transduced with MN1+HOXA9 proliferated slower than wildtype cells in response to both GM-CSF and IL3/IL6/SCF (p<.05). CRU assays with MN1+HOXA9-transduced Stat1 −/− and Stat5b −/− cells demonstrated that the day 6 CRU was 6 and 77 fold reduced, respectively, compared to wildtype cells. As MN1 and HOXA9 are upregulated in distinct subsets of normal karyotype AML we speculated that their combined overexpression may model subsets of complex karyotype AML. We performed gene set enrichment analysis on cytogenetic subsets of previously published gene expression data from 285 AML patients. 12 of 13 Stat-related pathways were enriched in complex karyotype patients compared to 4 and 8 of 13 Stat-related pathways in inv(16) and normal karyotype AML, respectively, thus supporting a critical role of Stat activation in LSCs of AML with multiple active pathways like complex karyotype AML. In conclusion we demonstrate considerable heterogeneity of LSC fidelity depending on the number of activated oncogenes and establish a critical role of Stat1 and Stat5b in mediating this LSC fidelity. Stat1 and Stat5b may become important therapeutic targets in complex karyotype AML.

Identification of New Markers Discriminating Between Myeloid and Lymphoid Acute Leukemia

The heterogeneity of acute myeloid leukemia (AML) with respect to biology and clinical course resides in the fact that patients belonging to the same group show marked differences in their response to chemotherapy, which would necessitate a refinement of AML classification. In order to contribute to define molecular markers for AML we realized microarray assays on two M5 AMLs and selected four differentially expressed genes to validate their expression by RQ-PCR. We have shown that two down-regulated genes in AML, Guanine nucleotide binding protein (G protein) gamma 11 (GNG11) and Amphiregulin are also down-regulated in B-ALL and T-ALL patients. We have found a gene, Ceruloplasmin, which is up-regulated in AML but not in B-ALL and T-ALL. The level of expression of these genes varies from one patient to another. Since the group of patients studied is limited, further studies must carry on with a larger series of patients to be able to make subdivision according to the expression of GNG11, Amphiregulin and Ceruloplasmin. Our study is the first to analyze these genes in AML, B-ALL, T-ALL and CL patients by quantitative real time PCR. This rapid and sensitive method could be used to screen these genes in different types of leukemia.

Gene Expression Profiling in AML with Normal Karyotype: A Multicenter Study Investigating Molecular Markers in 252 Cases

Acute myeloid leukemia (AML) is a heterogeneous disease and AML with normal karyotype (AML-NK) is a subgroup with intermediate prognosis. Over the past years molecular analyses have led to the identification of multiple molecular biomarkers that will further allow to dissecting clinically meaningful subgroups in this disease. Here, we present a multicenter study investigating whole-genome expression profiles of 252 cases of AML-NK. In three centers (Dresden, n=78; Munich, n=97; Ulm, n=77) Affymetrix HG-U133 Plus 2.0 microarray analyses were performed according to a standardized protocol. In a first series of analyses we focused on nucleophosmin gene (NPM1) mutations, the most common genetic lesion described in adult de novo AML to date. Gene expression signatures for 138 NPM1-mutated cases were compared to the profiles of 114 NPM1-unmutated cases. Supervised classification analyses resulted in >95% prediction accuracy of NPM1 mutation status (10-fold cross-validation). The sensitivity was very high for the positive detection of NPM1-mutated cases (>97%). Using a resampling approach (100 iterations) and splitting the complete data into a training set (n=168) and test set (n=84) the following genes were amongst the most frequently selected with higher expression in NPM1-mutated cases: HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB9, MEIS1, and PBX3. Lower expression in NPM1-mutated cases was observed for ABCB1, BAALC, MN1, MLLT3, or SPARC. Furthermore, our signature also showed a strong overlap to predictive NPM1 mutation signatures as published by Verhaak et al., and Alcalay et al., with 16/20 common genes and 15/18 common genes, respectively. Interestingly, some few NPM1-unmutated cases were repeatedly classified as being NPM1-mutated suggesting mutations in gene sequences not routinely screened. A strong HOXA-cluster signature is clearly dominant as well in these cases, whereas HOXB-cluster genes demonstrated a similar expression as compared to NPM1-unmutated cases. With respect to the robustness of differential gene expression signatures in NPM1 mutated cases across the laboratories using the top 300 differentially expressed genes, an overlap of 67 (22.3%) probe sets was observed between all three laboratories (NPM1-mutated vs. NPM1-unmutated: Dresden: 36 vs. 42; Munich: 42 vs. 55; Ulm: 36 vs. 41). In a second series of analyses we investigated cases with CCAAT/enhancer binding protein alpha (CEBPA) gene mutations. Gene expression signatures for 26 CEBPA-mutated cases were compared to the profiles of 132 CEBPA-unmutated cases. Supervised classification analyses resulted in >90% prediction accuracy of CEBPA mutation status (10-fold cross-validation) and frequently selected genes included HOXA2, HOXA3, HOXA5, HOXA7, HOXA9, HOXA10, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, and HOXB9. Thus, several HOX genes were overlapping to the NPM1 signature, albeit with an inverse correlation, i.e., HOX genes with relative overexpression expression in NPM1-mutated AML-NK demonstrated low expression in CEBPA-mutated cases. Finally, all 252 cases have also been characterized according to their fms-related tyrosine kinase 3 gene (FLT3) internal tandem duplication mutation status and this data is currently being integrated with the obtained signatures from above to develop a multi-gene prognostic model for AML-NK. In conclusion, our gene expression profiling study demonstrates robust signatures for AML-NK with NPM1 or CEBPA mutations. As our knowledge of this heterogeneous disease is increasing it seems possible to refine the classification of AML by incorporating molecular risk-based gene expression signatures.