AbstractAcute Myeloid Leukemia Patients Research Articles

Abstract 382: Inhibition of mitochondrial spare respiratory capacity by new securinine derivatives as a novel differentiation therapy for acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is characterized by a differentiation block in myeloid progenitors resulting in aberrant proliferation of immature myeloid cells. Therapeutics that induce differentiation of leukemic cells would overcome the high toxicity and low potency of conventional chemotherapy. Securinine, a plant-derived alkaloid from Securinega suffruticosa, has been shown to be a promising AML differentiation agent through an unclear mechanism, but is limited by its low potency (~20μM) and toxicity. We performed lead optimization chemistry on securinine that led to derivatives 250 and 317, with nanomolar efficacy and reduced toxicity. These derivatives effectively inhibit growth of AML cell lines and patient samples. Treatment of AML cell lines with compounds 250 and 317 induces the expression of differentiation markers CD11b and CD14c. Compounds 250 and 317 significantly reduced leukemic burden in a circulating model of AML in NSG mice. Additionally, these compounds were effective in inhibiting cell growth of HL60 cells that are resistant to a conventional chemotherapeutic, doxorubicin. By using the drug-affinity responsive target sensitivity (DARTS) assay, we identified that the redox mediator thioredoxin reductase (TrxR) is the primary target of these derivatives. 250 and 317 inhibit TrxR activity in vitro and in cells, and the resultant alterations in redox status are overcome by addition of a scavenger of reactive oxygen species (ROS), N-acetylcysteine. The ensuing oxidative metabolic stress rapidly alters the metabolite profile in AML cells including depletion of malate. Securinine derivatives decreased basal oxygen consumption rate and spare respiratory capacity, resulting in differentiation and cell death. Downregulating TrxR1 expression increased the sensitivity of OCI-AML3 cells to compounds 250 and 317. Together, we show here that these new derivatives of securinine exhibit preclinical efficacy in AML by decreasing mitochondrial spare respiratory capacity through the inhibition of TrxR. Citation Format: Sheela Karunanithi, Ruifu Liu, Anne Roe, Stephen Moreton, Yongchun Hou, Natasha Oldford, Juan Valentin Goyco, David Wald. Inhibition of mitochondrial spare respiratory capacity by new securinine derivatives as a novel differentiation therapy for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 382.

Abstract 1131: IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that occurs disproportionately in the elderly population. First line therapy is a regimen of anthracycline and cytarabine treatment or, more recently, demethylating agents. However, elderly AML patients are unable to tolerate aggressive chemotherapy regimens and there is an urgent need for targeted, tolerable therapies. To identify candidate pathways for targeted treatments, a microarray on transcripts for inflammatory signaling was performed on 588 primary patient AML samples. Non-hierarchical clustering revealed that Toll-like receptor (TLR) signaling transcripts are overexpressed in myelomonocytic and monocytic AML subtypes (M4 and M5 AML subtypes, respectively). Myeloid differentiation primary response 88 (MYD88) and downstream IL-1 receptor-associated kinases (IRAKs) were found to be overexpressed in these AML subtypes. MYD88 is an adaptor molecule for IL-1 and most TLR mediated signaling. To determine whether MYD88-dependent signaling is required for leukemogenesis, we derived a CreERT2-LoxP inducible system to delete Myd88 in a murine leukemia model. Because M4 and M5 AML subtypes are enriched for mutations in the mixed lineage leukemia (MLL) gene and overexpressed TLRs, MYD88, and IRAK2, an MLL-AF9 expressing construct was utilized to transform murine progenitor cells into leukemia cells. To study whether Myd88-dependent signaling is required for in vitro colony formation of the leukemic cells, Myd88 knockout (Myd88-/-) MLL-AF9 leukemic cells were subjected to colony-forming assays. Compared to Myd88 wild-type (Myd88fl/fl and Myd88+/+) leukemia cells, reduced colony-forming capacity was observed in the Myd88-/- leukemia cells. Further, reduced proliferation was observed in Myd88-/- cells compared to Myd88+/+ cells. Cell surface staining indicates Myd88-/- cells exhibit a loss of CD117, a marker of myeloid stemness, and a gain of CD11b, a marker of myeloid differentiation, when compared to Myd88+/+ cells, indicating that Myd88 deletion correlates with MLL-AF9 leukemia cell partial differentiation. In vivo transplantation of Myd88-/- cells delays leukemia development compared to Myd88+/+ cells. To determine whether IRAK1, a downstream kinase of MYD88, is also required to prevent partial differentiation, an IRAK1 shRNA knockdown cell line was generated and assayed for partial differentiation. In IRAK1KD MLL-AF9 cells, no partial differentiation was detected. Similarly, application of an IRAK1/4 inhibitor to Myd88+/+ MLL-AF9 cells failed to induce partial differentiation, despite suppressing proliferation. This indicates that MYD88-dependent signaling has functions independent of IRAK1 signaling that prevent partial differentiation in MLL-AF9 leukemia cells. Thus, MYD88 and IRAK1 signaling appear to mediate divergent functions, and both MYD88 and IRAK1 function appear to be required for MLL-AF9 leukemia. Citation Format: Joseph M. Cannova, Wei Wei, Rafael Gutierrez, Peter Breslin, Jiwang Zhang. IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1131.

Abstract 1890: Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease classified into 9 FAB subtypes according to the origin and/or maturity of the malignant cells: M0 undifferentiated, M1 minimally mature myeloblastic, M2 mature myeloblastic, M3 promyelocytic, M4 myelomonocytic, M4eos eosinophilic, M5 monocytic, M6 erythrocytic and M7 megakaryocytic. Overcoming this heterogeneity by the induction of a common surface antigen which would already be the target of an existing immunotherapy would be of great potential therapeutic interest to standardize a common immunotherapy approach to AML. Inecalcitol, a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential, has been shown to stimulate CD38 expression at the surface of the M3 subtype HL-60 cell line and of the M5 subtype U-937, MOLM-13 and THP-1 cell lines (ASH 2016, AACR 2017). We now report the effect of inecalcitol on AML cell lines representative of the 7 other FAB subtypes: KG-1 (M0), UOC-M1 (M1), Kasumi-1 (M2), OCI-AML2 (M4), EOL-1 (M4eos), HEL-92.1.7 (M6) and CMK (M7). Inecalcitol induced CD38 at the surface of CD38- cell lines (HL-60, U-937 and EOL-1) and strongly increased CD38 density at the surface of already CD38+ cell lines (KG-1, UOC-M1, Kasumi-1, OCI-AML2, MOLM-13, THP-1 and CMK) in a concentration-dependent manner, but remained without effect on CD38- HEL-92.1.7 cells. We have quantified the number of CD38 molecules at the surface of all cell lines (except HEL-92.1.7 which was unresponsive to inecalcitol) using the CellQuant calibrator (Biocytex) analysis by flow cytometry. In basal vehicle-treated condition, the number (in thousands, mean ± sem, n=2 or 3) of CD38 molecules per CD38+ cell were: Kasumi-1, 1.5 ± 1.0; MOLM-13, 2.5 ± 0.6; KG-1, 2.7 ± 0.9; THP-1, 7.0 ± 0.2; CMK, 12.1 ± 1.5; OCI-AML2, 16.1 ± 1.2. With 10nM inecalcitol, a maximal increase in CD38 density was usually obtained within 3 days, or after 7 days for KG-1 and CMK: Kasumi-1, 16.1 ± 3.3 (x11); MOLM-13, 43.3 ± 0.3 (x17); KG-1, 15.4 ± 3.7 (x5.7); THP-1, 17.9 ± 0.6 (x2.3); CMK, 15.4 ± 3.3 (x1.3); OCI-AML2, 73.5 ± 3.5 (x4.6). After 3 days of exposure to 10 nM inecalcitol, CD38 molecules appeared at the surface of initially CD38- cells and became quantifiable: U-937, 7.5 ± 0.4 (n=3); HL-60, 24.4 ± 4.9 (n=3) and EOL-1 ranged from 5.5 to 72.7 (n=2). In conclusion, after exposure to inecalcitol, CD38 appeared at the surface of CD38- AML cell lines and the density of CD38 increased at the surface of CD38+ AML cell lines, altogether representative of all 9 FAB classification subtypes M0 to M7, except M6 (erythrocytic) which represents 5% of all AML cases. These results suggest that treatment by inecalcitol may render 95% of AML patients sensitive to highly sensitive to anti-CD38 immunotherapy such as daratumumab. Citation Format: Enguerran Mouly, Cécile Planquette, Emilie Rousseau, Rémi Delansorne. Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1890.

Abstract 1787: COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive blood cancer with neoplastic infiltration of leukemic blasts in blood, bone marrow and viscera. Current treatment options have limited efficacy, and the 5-year survival rate is only 27%. CD3 bispecific antibodies that re-direct T cells towards the tumor have shown promising efficacy in hematological malignancies. The CD3/CD19 bispecific antibody blinatumomab has recently been approved by the FDA for the treatment of B-cell acute lymphocytic leukemia (ALL) (Kantarjian H et al (2017) NEJM). We evaluated COVA4231, a CD3/CD33 bispecific FynomAb, preclinically as therapeutic candidate for AML. CD33 is a cell surface receptor expressed on blasts of the majority of AML patients (Walter RB (2014) Expert Opin Ther Targets). COVA4231 was constructed by fusing the CD33-specific Fynomer D5 - a small (7 kDa) globular protein derived from the human Fyn SH3 domain with engineered affinity for CD33 - to a CD3-specific antibody with a novel silent IgG1 Fc. COVA4231 induced T cell activation and cytokine release in a CD33-dependent manner, and elicited potent T cell mediated cytotoxicity of CD33-expressing target cells in vitro with EC50 in the range of 4 - 29 pM (E:T cell ratio of 2:1, KG-1 or MOLM-13 as target cells). COVA4231 showed potent activity against 13 out of 15 primary AML blast samples ex vivo at E:T cell ratio of 1:1. The in vivo efficacy of COVA4231 was investigated in a subcutaneous HL-60 leukemia xenograft model in NSG mice in the presence of human T cells. COVA4231 was highly active across a wide dose range (0.05 - 5 mg/kg) and prevented tumor outgrowth in all treated mice. COVA4231 demonstrated an IgG1-like pharmacokinetic profile in mice with a terminal half-life 15.8 days (total drug), providing the opportunity to avoid continuous intravenous infusion protocols required for established CD3 bispecific formats (e.g. (scFv)2). In conclusion, COVA4231 is a highly active therapeutic candidate in vitro, in vivo and ex vivo, has IgG-like pharmacokinetics and is a promising therapeutic candidate for further preclinical and clinical development. Citation Format: Kristina Klupsch, Vanessa Baeriswyl, Roland Scholz, Joana Dannenberg, Roger Santimaria, David Senn, Elena Kage, Adrian Zumsteg, Isabella Attinger-Toller, Ulrike von der Bey, Susann König-Friedrich, Fanny Dupuy, Wibke Lembke, Clara Albani, Severin Wendelspiess, Lucijana Dinkel, Chelsea J. Gudgeon, Roland B. Walter, Julian Bertschinger, Simon Brack. COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1787.

Abstract 5681: Prediction of immunotherapy outcome by multimodal assessment of minimal residual disease and persistence of allogeneic anti-CD123 CAR T-cells (UCART123) in pre-clinical models of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a fatal disease. The promise of autologous T-cells expressing chimeric antigen receptors (CARs) in targeting B-cell malignancies has encouraged extension of this approach to AML. However, clinical guidance regarding infusion and re-infusion regimens for CAR-T technology is unclear for any cancer including AML. Given that studies demonstrate clear benefit for minimal residual disease (MRD) assessment in predicting relapse for AML, we thus sought to ascertain whether simultaneous molecular assessment of MRD markers and CAR-T-specific markers could inform decisions around CAR-T dosing and re-infusion. We tested this approach using patient-derived xenograft (PDX) models with allogeneic anti-CD123 CAR-T cells (UCART123). UCART123 are genetically modified allogeneic T-cells expressing an anti-CD123 CAR. These cells lack expression of the T-cell receptor (TCRabKO), in order to minimize graft vs. host disease (GvHD). PDX were established using prognostically adverse AML (FLT3-ITD+NPM1+) and treated with 1x106 or 2.5x106 UCART123. The median overall survival (OS) of control mice injected with saline or CAR-T negative (TCRabKO) T-cells succumbed to disease was 124.5 and 126 days, respectively. In contrast, UCART123 groups survived >180 days (hazard ratio 0.08, P=0.003). Clonal dynamics between disease and CAR-T were simultaneously monitored post-infusion by quantifying mutated NPM1 and CAR-T genetic markers, respectively, using digital droplet PCR (ddPCR). We found that ddPCR monitoring was more sensitive than multiparameter flow cytometry (MFC) at detecting MRD and persistence of UCART123. Using ddPCR, leukemia and UCART123 cells were detected when human cells were not evaluable using MFC in peripheral blood (PB). Mice with persistent UCART123 remained disease-free. Importantly, when mutated NPM1 levels became elevated with simultaneous loss of UCART123, relapse was evident by MFC in PB in subsequent time-points (2 out 20 mice, all at 1x106 dose, ~180 days) re-infusion of UCART123 cells resulted in effective elimination of AML. Taken together, we have demonstrated that simultaneous monitoring of disease and UCART123 cells provides valuable insight into the kinetics and effectiveness of UCART123 cells. Currently, we have implemented the ddPCR assay in the phase I clinical trial of UCART123 in AML allowing to simultaneously detect UCART123 cells and blasts in peripheral blood of NPM1 mutant AML patients. Citation Format: Mayumi Sugita, Nuria Mencia-Trinchant, Nathan Ewing-Crystal, Gabrielle Suppa, Roman Galetto, Agnès Gouble, Julianne Smith, Gail J. Roboz, Duane C. Hassane, Monica L. Guzman. Prediction of immunotherapy outcome by multimodal assessment of minimal residual disease and persistence of allogeneic anti-CD123 CAR T-cells (UCART123) in pre-clinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5681.

Abstract 1938: Store-operated calcium signaling is an effective therapeutic target in AML

Abstract Acute myeloid leukemia (AML) is the most prevalent adult leukemia characterized by genetic or mutational disruption of myeloid differentiation, growth arrest, and apoptosis. Standard “7 + 3” chemotherapy consisting of cytarabine in combination with an anthracycline such as daunorubicin has been used for more than four decades. However this course only results in 10 year disease free survival in 15% of patients age <60 years and 2% among those > 60 years. Several new successful therapies in AML are shown to work by promoting differentiation, and/or inducing apoptosis of the terminally differentiated cells. However these therapies, while promising, have the limitation of working in only a subset of AML with specific genetic profiles. The discovery of novel agents applicable to a broader patient population still represents a critical unmet need in this disease. Intracellular calcium is a common signaling molecule used in a variety of cellular processes including cell cycle progression. One of the major pathways which regulates calcium entry is store operated calcium entry (SOCE) mediated through calcium release-activated Ca2+ channels (CRAC), which respond to depleted calcium stores in ER lumen. This primarily occurs via interaction of ORAI1 (expressed on the plasma membrane) and STIM1 (expressed on the endoplasmic reticulum). Leukemic cells in particular often rely on the influx of calcium through CRAC for proliferation, therefore targeting these channels are a promising therapeutic option. In the current study, we explore the preclinical use of RP4010, a novel inhibitor of ORAI1 developed by Rhizen Pharmaceuticals, in AML. RP4010 is very effective at inhibiting proliferation of both AML cell lines and primary AML patient samples (IC50 values ranging from 0.2 - 1 μM using a tetrazolium-based colorimetric assay (MTS) assay). Importantly, RP4010 efficacy is not altered in the presence of stromal cell support. RP4010 also decreased colony growth in CFU assays. Finally, we evaluated the in vivo efficacy using an MV4-11 xenograft model. RP4010 treatment at 50 mg/kg once daily by oral gavage significantly prolonged survival compared to the vehicle control (p = 0.019). Furthermore, at the time of sacrifice, splenic tumor burden (percentage of human CD45 positive cells) was reduced in the mice treated with RP4010 compared to the vehicle group (p = 0.019). In conclusion, we provide here compelling evidence that inhibition of ORAI1 with RP4010 is a promising therapeutic strategy in AML. A Phase 1/1b study in R/R Non-Hodgkin Lymphoma (NHL) patients is currently underway in the US (ClinicalTrials.gov Identifier: NCT03119467), and our data would support expansion to other hematological malignancies as well, particularly AML. Senior authors J. C. B and E. H contributed equally to this work. Citation Format: Bridget Carmichael, Amy Lehman, Ola A. Elgamal, Shelley J. Orwick, Jean Truxall, Larry Beaver, Kumar V. Penmetsa, Srikant Viswanadha, John C. Byrd, Erin Hertlein. Store-operated calcium signaling is an effective therapeutic target in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1938.

MYB induces the expression of the oncogenic corepressor SKI in acute myeloid leukemia.

Acute myeloid leukemia (AML) arises through clonal expansion of transformed myeloid progenitor cells. The SKI proto-oncogene is highly upregulated in different solid tumors and leukemic cells, but little is known about its transcriptional regulation during leukemogenesis. MYB is an important hematopoietic transcription factor involved in proliferation as well as differentiation and upregulated in most human acute leukemias. Here, we find that MYB protein binds within the regulatory region of the SKI gene in AML cells. Reporter gene assays using MYB binding sites present in the SKI gene locus show MYB-dependent transcriptional activation. SiRNA-mediated depletion of MYB in leukemic cell lines reveals that MYB is crucial for SKI gene expression. Consistently, we observed a positive correlation of MYB and SKI expression in leukemic cell lines and in samples of AML patients. Moreover, MYB and SKI both were downregulated by treatment with histone deacetylase inhibitors. Strikingly, differentiation of AML cells induced by depletion of MYB is attenuated by overexpression of SKI. Our findings identify SKI as a novel MYB target gene, relevant for the MYB-induced differentiation block in leukemic cells.

Immune Checkpoints of CD8+ T Cells in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most common types of hematopoietic malignancy in the adults. Induction chemotherapy followed by post-remission is the general treatment for AML patients with high relapse rate and poor prognosis, which is often related to the CD8+ T cell immune status of the AML patients. Herein, we will make a mini review of phenotype-immune checkpoints expression and function of CD8+ T cells in AML based on our data and other groups, and this might indicate the application of immune checkpoints blockade alone or in combination in AML treatment.

Application of Molecular Karyotyping in Acute Myeloid Leukemia: A Review

Acute myeloid leukemia (AML) is an aggressive disease characterized by the overproduction of immature myeloid cells that accumulate in blood and bone marrow. Integration of genetic findings and clinicopathological information is crucial in establishing the diagnosis, prognosis and determining the therapeutic approach in the management of AML patients. In recent years, the AML classification has evolved from morphology to cytogenetics/molecular genetics-based findings, which is essential in the detection of chromosomal abnormalities and has provided the framework for the diagnosis and risk-stratification in AML. Moreover, with advances in molecular karyotyping such as comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays, various limitations of conventional diagnostic approaches have been overcome. Hence, this review focuses on the insights into molecular karyotyping using CGH and SNP arrays which enable the identification of copy number variations (CNVs) at a higher resolution and facilitate the detection of copy neutral loss of heterozygosity (CN-LOH) otherwise undetectable by conventional cytogenetics. Technical hindrances of these methods (e.g. regions of losses, gains, or “undulating waves”) are also discussed in the context of AML.

Prognosis of Patients with Acute Myeloid Leukemia Regarding the Presence FLT3 Gene Mutation – a Case Report

Abstract Acute myeloid leukemia (AML) is a cancerous disease affecting the myeloid line of the bone marrow cells. FLT3, also known as CD135, is a proto-oncogene, which, if mutated, leads to different types of cancer. The protein it encodes presents tyrosine-kinase activity, and its intratandem mutation, FLT3-ITD, leads to uncontrolled proliferation of myeloblasts and worse outcomes in AML patients. There are currently several pharmacological agents that can inhibit the effect of either the proteins with tyrosine-kinase activity or the mutated FLT3 gene. We present the case of a 68-year-old patient, smoker, with a history of arterial hypertension, chronic obstructive pulmonary disease, presenting with headache unresponsive to antalgics, dyspnea after physical exertion, and epistaxis, with onset 2 months prior to his presentation. The patient was diagnosed with AML with positive FTL3 mutation for which conventional induction therapy was initiated. Within the next days, the patient presented several complications related to the disease itself or caused by the treatment, which eventually led to his death.

JAM-C Identifies Src Family Kinase-Activated Leukemia-Initiating Cells and Predicts Poor Prognosis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here, we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation was uniquely found within the JAM-C-expressing LSC compartment. Cancer Res; 77(23); 6627-40. ©2017 AACR.

A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402-13. ©2017 AACR.

Immunomodulatory Drugs: IMiDs in Acute Myeloid Leukemia (AML).

AML patients have an aberrant and dysfunctional immune state, paving the way for novel agents targeting pathways that integrate with immune signaling, function, and response. Small molecule immunomodulatory drugs (IMiDs) represent a class of agents derived from the parent compound, thalidomide. There are currently 3 IMiDs approved for a variety of malignancies: thalidomide, lenalidomide, and the newest agent, pomalidomide. IMiDs lead to a multitude of immunobiologic effects such as cytokine modulation, co-stimulation of T cells, down-regulation of co-inhibitory molecules, enhancing natural killer cell activity, inhibition of regulatory T cells, and repairing perturbed synapse formation on T cells. IMiDs have been extensively studied in various AML settings with promising clinical activity. This review discusses the immunologic effects of IMiDs, the rationale for studying IMiDs in AML, and the published and ongoing clinical trials investigating IMiD activity in AML.

Molecular Study of NPM1-A (Nucleophosmin1-A) Mutation in Iraqi Adult Acute Myeloid Leukemia Patients; Its Correlation with Clinicopathological Parameters

Acute myeloid leukemia (AML) is a hematological malignancy of myeloid progenitor cells characterized by anomalous proliferation, inhibition of differentiation and expansion of leukemic cells blocked at the early stage of hematopoiesis. The molecular markers have become a smart tool to further division of AML patients into subgroups. Nucleophosmin (NPM1) mutations are found in approximately 30% of adult AML patients and are associated with a favorable outcome when detected in absence of FLT3-ITD mutation. Over 50 molecular NPM1 mutation variants have been identified; the most common one is NPM1-A mutation. The current study aims to detect; the frequency of NPM1-A in Iraqi adult newly diagnosed AML patients using real time – PCR technique and evaluate the relationship of NPM1-A with age, gender, total white blood cell (WBC) count and FAB subtypes of the disease. The frequency of NPM1-A mutation in newly diagnosed AML patients was 18.86%. Age was non-significantly higher in NPM1-A mutated patients than in patients without mutation (P=0.538). The NPM1-A mutation was predominantly seen in male (P= 0.069). The mutation was non-significantly higher among monocytic subtypes (M5+M4) compared with the other subtypes of the disease (P=0.916). In the NPM1 mutated group, the total WBC count was non-significantly higher than the non-mutated group (P=0.302). These findings suggest; Real time-PCR technique using TaqMan probe was very specific molecular test for this mutation. Mutated NPM1-A patients were associated with increased age, a higher total WBC count, male predominance and the monocytic subtypes of the disease.

Application of the AMLprofiler Diagnostic Microarray in the South African Setting.

Acute myeloid leukemia (AML) is characterized by proliferation of the myeloid lineage and accumulation of immature hematopoietic cells in the bone marrow and is typified by marked heterogeneity both in response to treatment and survival. AMLprofiler is a qualitative in vitro diagnostic microarray incorporating seven molecular biomarkers used to diagnose and predict posttherapy survival rates. In this study, we compared AMLprofiler to routine AML diagnostic methodologies employed in South Africa, focusing on consistency of the results, cost, and time to result. RNA was isolated from bone marrow and peripheral blood samples from patients with de novo AML and was processed using Affymetrix Gene Profiling Reagent kits. The results from AMLprofiler and standard methodologies were highly comparable. In addition, many samples were determined to be positive for biomarkers not routinely investigated in South Africa, namely, CEBPA double mutants, NPM1 variants, and altered expression levels of BAALC and EVI1. 38% of samples presented with no positive biomarker; AMLprofiler nonetheless enabled 26% of AML patients to be classified into either favorable or poor prognostic categories. This study highlights the comprehensive nature of the microarray. Decreased time to result and refinement of risk stratification are notable benefits.

Fitness in the elderly: how to make decisions regarding acute myeloid leukemia induction.

Acute myeloid leukemia (AML) is a disease of the elderly, but less than half of these patients are offered therapy despite the evidence of better survival with treatment in this patient population. Assessing fit, vulnerable, and frail older adults with AML remains a challenge for the treating oncologist. A majority of AML patients are elderly and often have significant comorbidities, lack of social support, and older caregivers. Performance status (PS), a subjective measure of how a patient will tolerate cancer chemotherapy, has been strongly correlated with mortality in older AML patients. However, a large portion of older adults have poor PS as a result of their underlying AML, and these patients may end up being undertreated. Conversely, some patients with excellent PS unexpectedly end up with excessive toxicity and mortality. The treating physician thus needs a more objective and comprehensive method to differentiate patients along the fit-frail spectrum irrespective of their chronological age. For more than a decade, comprehensive geriatric assessment has been shown to improve routine oncology assessment by adding information about the functional, emotional, cognitive, and social status of older patients with cancer. In addition to the chronological and functional age, there is an attempt to quantify a patient's biological age to aid in better decision making. This chapter attempts to review the clinical challenges of AML treatment in the elderly population and to highlight the current literature and future research required to be able to assess fitness and maximize therapeutic options in this heterogeneous patient population.

Eradication of Acute Myeloid Leukemia with FLT3 Ligand-Targeted miR-150 Nanoparticles.

Acute myeloid leukemia (AML) is a common and fatal form of hematopoietic malignancy. Overexpression and/or mutations of FLT3 have been shown to occur in the majority of cases of AML. Our analysis of a large-scale AML patient cohort (N = 562) indicates that FLT3 is particularly highly expressed in some subtypes of AML, such as AML with t(11q23)/MLL-rearrangements or FLT3-ITD. Such AML subtypes are known to be associated with unfavorable prognosis. To treat FLT3-overexpressing AML, we developed a novel targeted nanoparticle system: FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) nanosized dendriplex encapsulating miR-150, a pivotal tumor suppressor and negative regulator of FLT3 We show that the FLT3L-guided miR-150 nanoparticles selectively and efficiently target FLT3-overexpressing AML cells and significantly inhibit viability/growth and promote apoptosis of the AML cells. Our proof-of-concept animal model studies demonstrate that the FLT3L-guided miR-150 nanoparticles tend to concentrate in bone marrow, and significantly inhibit progression of FLT3-overexpressing AML in vivo, while exhibiting no obvious side effects on normal hematopoiesis. Collectively, we have developed a novel targeted therapeutic strategy, using FLT3L-guided miR-150-based nanoparticles, to treat FLT3-overexpressing AML with high efficacy and minimal side effects. Cancer Res; 76(15); 4470-80. ©2016 AACR.

Abstract 4696: The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia

Abstract Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients. Citation Format: Min Ye, Mike Liu, Jin Lu, Yvonne Lo, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Paul Waeltz, Melissa Ann Fischer, Chunhong He, Liangxing Wu, Xiaochuan Shan, Hong Chang, Maryanne Covington, Yanlong Li, Tim Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Michael Savona, Martin Carroll, Bruce Ruggeri, Sang Hyun Lee. The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4696.

Frequency and Dynamics of Leukemia-Initiating Cells during Short-term Ex Vivo Culture Informs Outcomes in Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LIC) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in vivo The xenotransplantation model captures functional properties of LICs that have clinical prognostic value. However, the long duration of this in vivo assay has hampered its use as a prognostic tool. Here, we show, using an ex vivo coculture system, that intermediate and poor risk AML patient samples at diagnosis have a 5 to 7 times higher frequency of leukemic long-term culture-initiating cells (L-LTC-IC) compared with the good risk group. We defined a fluorescence dilution factor (FDF) parameter that monitors sample proliferation over 1 week and established a strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor prognostic AMLs or for samples capable of engrafting NSG mice compared with good risk AMLs or nonengrafters. Importantly, FDF could classify normal karyotype intermediate risk patients into two groups with a significant difference in their overall survival, thus making this nongenetic and non-in vivo approach a new clinically relevant tool for better diagnosis of AML patients. Cancer Res; 76(8); 2082-6. ©2016 AACR.

Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Recent genome-scale sequencing efforts have determined that on average, an individual AML case is associated with 5 somatic mutations in recurrently mutated genes. This finding raises the important question of how AML develops from normal hematopoietic stem and progenitor cells. Given that AML is characterized by the sequential acquisition of genetic lesions in a single lineage of cells, and that all cells in the myeloid lineage, apart from HSC, are short-lived, we proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. We investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual HSC. Using exome sequencing, we defined mutations present in individual AML genomes from 19 cases, and screened for these mutations in the residual HSC. We identified multiple mutations present in residual HSC retaining normal multilineage differentiation in vivo, including mutations in IDH1/2, TET2, DNMT3A, and genes encoding the subunits of the cohesin complex. Through single cell analysis, we determined that as we hypothesized, a clonal progression of multiple mutations occurs in HSC. From these studies, we identified patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, while mutations in "proliferative" genes such as FLT3 and KRAS occur late. Using this approach, we identified pre-leukemic HSC in a larger cohort of AML patients, and determined that their frequency within the stem cell compartment at the time of diagnosis varied widely from undetectable to nearly 100% of the cells. Stratifying these patients into two groups with either high or low frequencies of pre-leukemic HSC demonstrated that patients in the high group had much worse overall and relapse-free survival than those in the low group, indicating that the presence of pre-leukemic HSC may be critical for eventual clinical outcomes. To further investigate the response of pre-leukemic HSC to treatment, we analyzed the persistence of pre-leukemic mutations in patients in remission and found CD34+ progenitor cells and various mature cells that harbor pre-leukemic mutations. These findings indicate that pre-leukemic HSC can survive induction chemotherapy, identifying these cells as a potential reservoir for the re-evolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease, and show that some of these patterns are consistent with involvement of pre-leukemic HSC. Thus, our studies of pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations, suggest a potential mechanism contributing to relapse, and constitute a cellular reservoir that may need to be targeted for more durable remissions. Disclosures Majeti: Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.