Leukemia Fusion Research Articles

An Orally Bioavailable and Highly Efficacious Inhibitor of CDK9/FLT3 for the Treatment of Acute Myeloid Leukemia.

Simple SummaryCDDD11-8 is an orally bioactive pharmacological inhibitor of CDK9. In AML cells, CDDD11-8 suppressed the proliferation and caused robust inhibition of tumor growth in vivo via synergistic inhibition of FLT3. The drug candidate has potential to overcome cancer cell resistance to FLT3 inhibition by concurrently blocking the CDK9-mediated upregulation of cancer cell-survival genes.Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in approximately one-third of AML patients and are associated with a particularly poor prognosis. The most common mutation, FLT3-ITD, is a self-activating internal tandem duplication (ITD) in the FLT3 juxtamembrane domain. Many FLT3 inhibitors have shown encouraging results in clinical trials, but the rapid emergence of resistance has severely limited sustainable efficacy. Co-targeting of CDK9 and FLT3 is a promising two-pronged strategy to overcome resistance as the former plays a role in the transcription of cancer cell-survival genes. Most prominently, MCL-1 is known to be associated with AML tumorigenesis and drug resistance and can be down-regulated by CDK9 inhibition. We have developed CDDD11-8 as a potent CDK9 inhibitor co-targeting FLT3-ITD with Ki values of 8 and 13 nM, respectively. The kinome selectivity has been confirmed when the compound was tested in a panel of 369 human kinases. CDDD11-8 displayed antiproliferative activity against leukemia cell lines, and particularly potent effects were observed against MV4-11 and MOLM-13 cells, which are known to harbor the FLT3-ITD mutation and mixed lineage leukemia (MLL) fusion proteins. The mode of action was consistent with inhibition of CDK9 and FLT3-ITD. Most importantly, CDDD11-8 caused a robust tumor growth inhibition by oral administration in animal xenografts. At 125 mg/kg, CDDD11-8 induced tumor regression, and this was translated to an improved survival of animals. The study demonstrates the potential of CDDD11-8 towards the future development of a novel AML treatment.

SIRT2 regulates proliferation and chemotherapy response of MLL-ENL-driven acute myeloid leukemia

Both MLL-AF9 and MLL-ENL leukemia fusion proteins drive oncogenic transformation of hematopoietic cells through their N-terminal DNA/histone binding mixed-lineage leukemia 1 domain and C-terminal fragment of AF9 or ENL containing an unstructured linker region and the ANC1 homology domain, which recruits transcription factors. Despite of their structural similarity, acute myeloid leukemia (AML) patients bearing MLL-ENL show more adverse outcomes compared to those with MLL-AF9. We recapitulated the clinical patterns of these two MLL-fusions driven AMLs using murine models and found that MLL-ENL AML cells showed slower cell cycle progression and more resistance to standard chemotherapy than MLL-AF9 cells. These phenotypes were primarily controlled by the linker regions of ENL and a highly conserved lysine residue K469 within. Substitution of K469 with an acetylated mimic glutamine abolished the ability of MLL-ENL to suppress proliferation and promote chemo-resistance. We showed that deacetylase Sirt2 might act as an upstream regulator of MLL-ENL. Deletion of Sirt2 promoted proliferation of AML cells with either MLL fusions. Importantly, loss of Sirt2 greatly enhanced the sensitivity of the MLL-ENL AML cells to chemo-treatment. Taken together, our study uncovered a unique regulatory role of Sirt2 in leukemogenesis and suggested targeting SIRT2 as a new way to sensitize MLL-ENL AML patience for chemotherapy.

HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis.

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

The Oncogenic Fusion Protein CBFB-SMMHC Downregulates CD48 to Evade NK Cell Recognition

Chromosomal translocations are a common cytogenetic aberration in acute leukemia, which frequently generate fusion proteins with oncogenic properties. We have previously studied the immune evasion mechanisms of two common oncogenic fusion proteins in acute myeloid leukemia (AML), PML-RARA and AML1-ETO. We investigated, in particular, the activity of natural killer (NK) cells, which are part of the innate immune system and play a significant role in eliminating leukemic cells. We found that both oncogenic fusion proteins downregulate the expression of CD48, a ligand of the NK cell activating receptor 2B4, to escape NK cell-mediated recognition of the tumor cells.In the current study we tested the possibility that other oncogenic fusion proteins are also able to evade NK cell recognition. To this aim we cloned several leukemic fusion proteins into lentiviral vectors and expressed them in U937 cells, which are commonly used to express leukemic fusion proteins. The leukemic fusion proteins included MLL-AF4, NUP98-HOXA9, DEK-NUP98 and CBFB-SMMHC. By using flow cytometry, we examined the effect of these fusion proteins on the expression of several NK cell ligands. CBFB-SMMHC was the only fusion protein which downregulated the expression of CD48.Next, we tested the functional significance of the downregulation of CD48 by CBFB-SMMHC by performing cytotoxicity assays with NK cells. We first used the NK cell line YTS eco since the cytotoxicity of these cells is mainly dependent on the 2B4-CD48 interaction. We found that cells which express CBFB-SMMHC were killed significantly less than the control cells, and that this reduced killing was due to CD48 downregulation. We also tested the killing of CBFB-SMMHC expressing cells by primary bulk NK cells and found that cells which express this fusion protein were killed significantly less.Mechanistically we show that overexpression of CBFB-SMMHC reduces the mRNA levels of CD48. Additionally, we tested whether treatment with HDAC inhibitors (HDACi) can reverse the downregulation of CD48 by CBFB-SMMHC. We treated cells which express CBFB-SMMHC with various HDACi and examined the effect of this treatment on the expression of CD48. Two specific class I HDACi, mocetinostat and entinostat, upregulated the expression of CD48 in cells which express this fusion protein.Taken together we show that modulation of CD48 expression is an immune evasion mechanism, which is specific to core binding factor (CBF) proteins (AML1-ETO and CBFB-SMMHC) and PML-RARA. Although the three fusion proteins which we found to down-regulate the expression of CD48 are associated with better prognosis in AML, about 30% of CBF-AML patients eventually relapse. Based on our findings we suggest that down-regulation of CD48 by these fusion proteins, which leads to NK cell immune evasion, contributes to the persistence of residual disease in these subtypes of leukemia and eventually to a clinical relapse. Finally, we propose that NK cell based therapies (i.e., NK cell infusion) or class I HDACi could be potential adjunctive therapies for CBF-AML or APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypomethylating Agent Leads to Enhanced Immunogenicity of a Dendritic Cell/Acute Myeloid Leukemia Fusion Vaccine and Prolonged Survival in an Immunocompetent Mouse Model

Immunotherapy for acute myeloid leukemia (AML) holds great promise as a modality to overcome tolerance and elicit memory responses needed for durable remissions. Our group pioneered a novel personalized vaccine that targets a broad array of leukemic antigens (Ags) by fusing whole patient-derived AML cells to autologous dendritic cells (DCs). In a clinical trial of AML patients in chemotherapy-induced remission, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Enhancing response to vaccination relies on increasing Ag presentation and overcoming factors in the microenvironment that contribute to tolerance.While the mechanism of action of hypomethylating agents (HMAs) is multifactorial, HMAs are increasingly recognized as exhibiting immunomodulatory functions thought to contribute to therapeutic efficacy. We have previously shown that the HMA SGI-110 augments AML Ag processing and increases AML susceptibility to T cell-mediated killing. In the present study, we aimed to elucidate the effect of HMAs on enhancing Ag presentation and modulating the AML microenvironment as well as to investigate the impact of HMA on the immunogenicity of a DC/AML vaccine.We first investigated the ability of SGI-110 to reverse critical aspects of the AML immunosuppressive milieu and enhance vaccine efficacy. C57BL/6J mice were retro-orbitally inoculated with 1x105 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1mg/kg) to assess immunologic effects without preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). SGI-110 therapy was also associated with decreased myeloid-derived suppressor cell (MDSC) burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control.Consistent with these findings, treated mice demonstrated increased AML-specific immunity via increased T cells isolated from the BM or spleen that expressed intracellular IFNϒ following ex-vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02).We then examined the potential synergy of SGI-110 + vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNϒ in response to tumor lysate as compared to vaccine or SGI-110 alone.To further elucidate the mechanism behind the immune activating effects of HMAs, we investigated the effect of HMA on the endogenous retroviral (ERV) pathway. Activation of this pathway results in increased IFN ß expression leading to increased MHC HLA Class I expression and subsequently, immune stimulation in a solid tumor model (Chiappinelli et al. Cell . 2015). MOLM-14 cells were exposed to low-dose HMA daily X 3 days and analyzed for ERV-3 expression using western blotting (WB). The results showed significant upregulation of ERV-3 expression after treatment. Due to activation of the ERV pathway, increased IFN ß expression was observed with WB analysis. Indeed, analysis of an HLA-A2 positive human AML cell line, THP1, showed significant HLA-A2 upregulation after HMA exposure. Most importantly, HLA-A2 upregulation led to a significant rise in tumor Ag presentation, detected by measuring the binding capacity of a unique T-cell receptor-like antibody using flow cytometric analysis in THP-1 cells.Given these effects, we sought to examine whether treatment with vaccine + SGI-110 would enhance survival in this model. Mice were treated with: vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a survival advantage compared to untreated mice (p<0.05). Notably, the combination arm further prolonged survival as compared to untreated mice (p<0.05).In conclusion, SGI-110 modulates critical factors contributing to the immunosuppressive milieu in AML by decreasing T cell PD-1 expression and promoting MDSC expansion, resulting in enhanced anti-AML immunity. Moreover, SGI-110 augments the immune response to an autologous DC/AML vaccine in a murine model resulting in a survival benefit. In addition, HMA enhances Ag presentation, in part due to activation of the ERV pathway. The combination of a novel personalized DC/AML fusion vaccine and HMA holds great potential and a clinical trial is being initiated. DisclosuresAvigan:Genus Oncology: Research Funding.

ETO2 Regulates Cell-Fate Genes and Controls Relapse in Acute Myeloid Leukemia

The cell-fate of hematopoietic stem cells (HSCs) and their malignant version, leukemia stem cells (LSCs), are controlled by antagonistic functions of two classes of genes. We defined these genes as pro-HSC/LSC and anti-HSC/LSC genes based on their abilities to increase or decrease the steady state numbers of stem cells. Expansion of LSCs is thought to be an early step towards relapse (Lapidot, Sirard et al. 1994, Huntly and Gilliland 2005, Vasanthakumar and Godley 2014), a medical condition with no effective cures. We show here that the expression of both pro- and anti-HSC/LSC genes are coordinately controlled by ETO2, a transcriptional corepressor of E-protein family of transcription factors (Zhang, Kalkum et al. 2004, Gow, Guo et al. 2014). In both HSCs and acute myeloid leukemia (AML) cells, ETO2 occupies active chromatin regions that encompass enhancers and promoters of pro- and anti-HSC/LSC genes. ETO2 orchestrates the binding of epigenetic modifiers and transcription factors to enhance the expression of self-renewal and survival genes while reducing the expression of growth arrest and pro-inflammatory genes. Further supporting a role of ETO2 in maintaining the self-renewable population of HSCs and LSCs, depleting ETO2 compromises self-renewal of HSCs and diminishes in vivo propagation of AML cells.Analysis of multiple patient datasets supports the conclusion that relapse requires a threshold level of ETO2 expression. Attaining this threshold level requires a conserved gene activation mechanism mediated by GCN5 histone acetyltransferase. Inhibiting GCN5 reduces ETO2 and abolishes colony-formation capacity of primary AML cells. We also show that the conserved and diverged functions between ETO2 and AML1-ETO, a leukemia fusion protein expressed in t(8;21) AML, combined with a newly-discovered ability of AML1-ETO to repress ETO2 transcription, defines the unique properties of t(8;21) AML and explains its long-observed favorable prognosis. Importantly, we show that despite an enriched expression of HSC/LSC genes, relapse is paused in t(8;21) AML due to AML1-ETO-mediated downregulation of ETO2 and requires overcoming this repression to proceed. Thus, targeting GCN5/ETO2 axis to achieve a similar effect as that imposed by AML1-ETO is expected to dramatically improve therapeutic efficacy of AML, whose current survival rate is worse than most cancers and leukemias (Supported by NIH grants R01HL093195-01A1 and R21CA178513 to JZ and T32 training grant T32GM008306-26A1 to NS).Gow, C. H., C. Guo, D. Wang, Q. Hu and J. Zhang (2014). “Differential involvement of E2A-corepressor interactions in distinct leukemogenic pathways.” Nucleic Acids Res 42(1): 137-152.Huntly, B. J. and D. G. Gilliland (2005). “Leukaemia stem cells and the evolution of cancer-stem-cell research.” Nat Rev Cancer 5(4): 311-321.Lapidot, T., C. Sirard, J. Vormoor, B. Murdoch, T. Hoang, J. Caceres-Cortes, M. Minden, B. Paterson, M. A. Caligiuri and J. E. Dick (1994). “A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.” Nature 367(6464): 645-648.Vasanthakumar, A. and L. A. Godley (2014). “On the origin of leukemic species.” Cell Stem Cell 14(4): 421-422.Zhang, J., M. Kalkum, S. Yamamura, B. T. Chait and R. G. Roeder (2004). “E protein silencing by the leukemogenic AML1-ETO fusion protein.” Science 305(5688): 1286-1289. DisclosuresNo relevant conflicts of interest to declare.

Potential Role of Ubiquitin E3 Ligase, FBXO9, As a Tumor Suppressor in AML

Hematopoietic stem cells (HSCs) give rise to all cells of the blood. The balance between HSC self-renewal and differentiation is critical to maintaining hematopoiesis throughout the life of the organism, and disruptions to this balance often result in malignancies, including acute myeloid leukemia (AML). Multiple transcription factor networks are known to be important regulators of HSCs and, when mutated, can lead to leukemia development. More recently, protein degradation by the ubiquitin proteasome system (UPS) has been shown to regulate HSC self-renewal, differentiation, and survival. The role of this pathway in leukemogenesis is less clear.We recently performed a screen in embryonic stem cells to identify members of the UPS involved in cell fate decisions. From this, we identified the Ubiquitin E3 ligase FBXO9, which is a substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex. Interestingly, FBXO9 is highly expressed in hematopoietic stem and progenitor cells, but is decreased in patients with AML including patients with Inv(16) chromosomal abnormality. In order to study FBXO9 in normal and malignant hematopoiesis, we utilized a novel method using CRISPR/Cas9 to generate a conditional knockout model in collaboration with the Mouse Transgenic Core at UNMC. We bred our mice to transgenic mice expressing Cre Recombinase from the Mx1 promoter, which allows for PolyIC inducible expression of Cre in all hematopoietic cells including HSC. Our preliminary data shows alterations in hematopoiesis, including less competitive repopulation capability of the HSC in vivo and decreased differentiation potential of the hematopoietic progenitor population (HPC). To test if Fbxo9 plays a role in leukemia development, we crossed mice with the conditional allele of Fbxo9 with a knockin mouse model of AML that expresses the leukemia fusion gene Cbfb-MYH11 . In a small cohort of mice, we found that deletion of Fbxo9 in the context of Cbfb-MYH11 caused markedly accelerated leukemia development with >50% myeloid blasts in the peripheral blood by week 6. These findings suggest that the ubiquitin E3 ligase, Fbxo9, is a novel tumor suppressor and can regulate self-renewal, differentiation, and malignant transformation. In addition The dynamic reversibility of the ubiquitin modification (by kinases, phosphatases, E3 ligases, and de-ubiquitinases) and the recent development of both general proteasome inhibitors as well as specific antagonists of ligase function suggest that manipulation of proteins by ubiquitin E3 ligases could provide potential targets for drug discovery and stem cell therapy. DisclosuresNo relevant conflicts of interest to declare.

A New Sensitive and Fast Molecular Analysis Method for the Identification of 30 Different Markers in Acute Myeloid and Lymphoid Leukemias at Diagnosis Time

Background: Medical advances in the treatment of Acute and Chronic Leukemia in the last decade were exciting, thanks to the discovery of new drugs and transplantation of hematopoietic stem cells; but so that these weapons can be used in an optimal way, it is necessary that the leukemia is diagnosed as quickly as possible and accurately to limit the spread of leukemia cells in the patient. The ability to detect fusion genes characteristics of these diseases has become essential but laborious, time consuming and generally comprises karyotype analysis, followed by FISH (Fluorescent in situ hybridization) and molecular analysis (RT-PCR). New molecular diagnostic technologies are being developed that can identify disease markers with extreme accuracy in a few hours (2-3 hours). The Leukemia Fusion Genes (Q30) Screening Kit (QuanDx, San Jose, CA 95131 USA; Distr. Resnova S.r.l.) is a real-time multiplex RT-qPCR based assay for detection of leukemia associated fusion gene transcripts in total RNA from bone marrow or whole blood samples, it is a qualitative test for the simultaneous detection of 30 characteristic fusion genes of acute leukemias. For Acute Myeloid Leukemia (AML) transcripts we can expect tested 18 fusion genes with 72 different breakpoints: BCR-ABL1, MLL-AF9, CBFbeta-MYH11, PML-RARalpha, AML-MDS1, NPM-MLF1, AML-ETO, NPM-RARalpha, PLZF-RARalpha, DEK-CAN, MLL-ELL, AML1-EAP, MLL-AF10, ST-CAN, TEL-ABL1, TLS-ERG, FIP1L1-PDGFR alpha, TEL-PDGFRbeta. For Acute Lymphoid Leukemia (ALL) transcripts we will detected 15 fusion genes with 71 different breakpoints: MLL-AF4, TEL-AML1, MLL-ENL, E2APBX1, BCR/ABL1, SIL-TAL1, E2A-HLF, MLL-AF6, CALM-AF10, HOX11, HOX11L2, MLL-AF10, HOX11, HOX11L2, MLL-AF10, SET-CAN, TEL-ABL1, TLS-ERG.Aim: The aim of this study is to evaluate and to standardize a new commercial kit, the Leukemia Fusion Genes (Q30) Screening Kit, that is reliable, reproducible and able to detect simultaneously, in one real-time PCR reaction and in a short time different rearrangement involved in acute leukemia at diagnosis.Methods: 60 samples will be analyzed as follows: 51 AML and 9 ALL.All samples have been previously tested with classical PCR qualitative methods (Leukemia 1999). 55 RNAs were extracted using the Maxwell 16 Semi-automatic Extractor (Simply RNA Blood kit) from isolated cells from Ficoll or buffycoat of bone marrow or peripheral blood. 5 RNAs were extracted with manual phenol-chloroform method. Concentration was determined by spectrophotometric reading (from 20ng/µL to 200ng/µL). cDNA was added to 8 qPCR reaction tubes, which contain specific PCR primers and probes for detection of fusion genes and an internal control gene (GUSB). The qPCR is performed in a real-time thermal cycler, the kit is compatible with the ABI7500 system.Results: 21 out of 51 AML patients (pts), tested with routine approach, had no markers, the remaining 30 AML had rearrangements and mutations as follows:1 pt CBFβ-MYH11, 2 pts PML-RARalpha, 2 pts AML1-ETO, 1 pt DEK-CAN, 4 pts FLT3 ITD, 6 pts NPM, 8 pts FLT3/NPM, 1 pt DEK-CAN/FLT3, 3 pts PML-RARα/FLT3 ITD, 1 pt IDH mut, 1 pt MLL ITD. 5/9 ALL samples had no markers, the remaining presented with t (4;11) (2 pts), t (9;22) (2 pts).All the tested samples (60 samples) confirmed the molecular rearrangement where it was present (14 samples) using primer sets for molecular rearrangements by RT-PCR technique. 100% concordance, 14 samples out of 14.Significantly 5 out of 60 patients showed new (previously unidentified) rearrangements with the primer sets used by the laboratory routinely: 2 pts with MLL-ELL/t(11;19), 1 pt with MLL-AF10/t(10;11) and 2 pts with AML1-MTG16/t(16;21). The kit revealed a percentage of rearranged patients equal to 32% compared to 23% analyzed with the primer sets used routinely, using a time of two/three hours against 72 hours, considering the totality of the rearrangements.Conclusion: The Q-Fusion system provides a sensitive instrument specific, reliable and cost effective, easy to perform for routine screening in leukemias at diagnosis.The kit can detect 30 common fusion genes, it can only report the types of fusion genes, but cannot differentiate the splice variants. It is a Real-Time multiplex RT-qPCR and the Cq values cannot be used for quantification of the fusion transcript level. DisclosuresSaglio:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria.

Discovery of DDO-2213 as a Potent and Orally Bioavailable Inhibitor of the WDR5-Mixed Lineage Leukemia 1 Protein-Protein Interaction for the Treatment of MLL Fusion Leukemia.

WD repeat-containing protein 5 (WDR5) is essential for the stability and methyltransferase activity of the mixed lineage leukemia 1 (MLL1) complex. Dysregulation of the MLL1 gene is associated with human acute leukemias, and the direct disruption of the WDR5-MLL1 protein-protein interaction (PPI) is emerging as an alternative strategy for MLL-rearranged cancers. Here, we represent a new aniline pyrimidine scaffold for WDR5-MLL1 inhibitors. A comprehensive structure-activity analysis identified a potent inhibitor 63 (DDO-2213), with an IC50 of 29 nM in a competitive fluorescence polarization assay and a Kd value of 72.9 nM for the WDR5 protein. Compound 63 selectively inhibited MLL histone methyltransferase activity and the proliferation of MLL translocation-harboring cells. Furthermore, 63 displayed good pharmacokinetic properties and suppressed the growth of MV4-11 xenograft tumors in mice after oral administration, first verifying the in vivo efficacy of targeting the WDR5-MLL1 PPI by small molecules.

Core-binding factor leukemia hijacks the T-cell–prone PU.1 antisense promoter

AbstractThe blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFβ-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.

A first-in-human phase I study of ATR inhibitor M1774 in patients with solid tumors.

TPS3153 Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase plays a critical role in the DNA damage response by sensing and responding to DNA replication stress, and by inducing cell cycle arrest to prevent aberrant replication and mitotic catastrophe. Based on extensive preclinical and limited clinical evidence, ATR inhibition is a promising treatment strategy as monotherapy for patients with advanced tumors harboring synthetically lethal conditions, such as alternative lengthening of telomeres (ALT) and inactivating mutations in ARID1A and ATM. M1774 is a potent, selective, orally administered ATR inhibitor that has been shown to exert antitumor activity in patient-derived xenograft tumors and acute myeloid leukemia xenograft tumors that express the ATR inhibition sensitizing mixed lineage leukemia fusion protein. This study (NCT04170153) aims to evaluate the safety and tolerability, maximum tolerated dose, recommended dose for expansion (RDE) and pharmacokinetics (PK) of M1774 (part A1), the effect of food on M1774 PK (part A2), and the efficacy of M1774 in patients with tumors harboring selected mutations (part A3). An additional objective is to assess the pharmacodynamics of M1774 by measuring relative changes in baseline p-CHK1 and γ-H2AX expression in paired tumor biopsies and serial blood samples. Methods: Patients aged ≥18 years, with an Eastern Cooperative Oncology Group performance status ≤1, adequate baseline hematological, renal and hepatic function, and with locally advanced or metastatic disease refractory to standard therapy are eligible. Patients with tumors bearing loss-of-function (LoF) mutations (determined by site testing or a central trial assay) in ARID1A, ATM, or ATRX and/or DAXX as ALT status surrogate markers; and measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, will be enrolled in part A3. In the dose escalation phase (part A1 [open]), 18–24 patients are due to receive M1774 at a starting dose of 5 mg once daily. Dose escalation is determined by the safety monitoring committee and guided by a Bayesian 2-parameter logistic regression model. The preliminary food assessment (part A2) will follow a randomized two-sequence two-period crossover design in which ≤12 patients will be randomized (1:1) to receive a single dose of M1774 on Day –7 at the RDE (determined in part A1) in either a fed or fasted condition. After the food assessment, patients will subsequently receive M1774 according to the part A1 dosing schedule. In the preliminary efficacy study (part A3), patients (n = 20–24 for each of the three planned cohorts) with tumors harboring LoF mutations in the genes for ARID1A, ATM, ATRX and/or DAXX, will receive M1774 at the RDE. The primary efficacy endpoint is overall response (RECIST). The study is open and currently recruiting. Patients have been enrolled to seven cohorts in part A1 with no DLTs observed; dose escalation is ongoing. Clinical trial information: NCT04170153.

CRLF2 and IKZF1 abnormalities in Mexican children with acute lymphoblastic leukemia and recurrent gene fusions: exploring surrogate markers of signaling pathways.

The gene fusions BCR‐ABL1, TCF3‐PBX1, and ETV6‐RUNX1 are recurrent in B‐cell acute lymphoblastic leukemia (B‐ALL) and are found with low frequency in coexistence with CRLF2 (cytokine receptor‐like factor 2) rearrangements and overexpression. There is limited information regarding the CRLF2 abnormalities and dominant‐negative IKZF1 isoforms associated with surrogate markers of Jak2, ABL, and Ras signaling pathways. To assess this, we evaluated 24 Mexican children with B‐ALL positive for recurrent gene fusions at diagnosis. We found CRLF2 rearrangements and/or overexpression, dominant‐negative IKZF1 isoforms, and surrogate phosphorylated markers of signaling pathways coexisting with recurrent gene fusions. All the BCR‐ABL1 patients expressed CRLF2 and were positive for pCrkl (ABL); most of them were also positive for pStat5 (Jak2/Stat5) and negative for pErk (Ras). TCF3‐PBX1 patients with CRLF2 abnormalities were positive for pStat5, most of them were also positive for pCrkl, and two patients were also positive for pErk. One patient with ETV6‐RUNX1 and intracellular CRLF2 protein expressed pCrkl. In some cases, the activated signaling pathways were reverted in vitro by specific inhibitors. We further analyzed a TCF3‐PBX1 patient at relapse, identifying a clone with the recurrent gene fusion, P2RY8‐CRLF2, rearrangement, and phosphorylation of the three surrogate markers that we studied. These results agree with the previous reports regarding resistance to treatment observed in patients with recurrent gene fusions and coexisting CRLF2 gene abnormalities. A marker phosphorylation signature was identified in BCR‐ABL1 and TCF3‐PBX1 patients. To obtain useful information for the assessment of treatment in B‐ALL patients with recurrent gene fusions, we suggest that they should be evaluated at diagnosis for CRLF2 gene abnormalities and dominant‐negative IKZF1 isoforms, in addition to the analyses of activation and inhibition of signaling pathways.

Tet1 is not required for myeloid leukemogenesis by MLL-ENL in novel mouse models.

The Ten Eleven Translocation 1 (TET1) gene encodes an epigenetic modifying molecule that is involved in demethylation of 5-methylcytosine. In hematological malignancies, loss-of-function mutations of TET2, which is one of the TET family genes including TET1, are frequently found, while the mutations of TET1 are not. However, clinical studies have revealed that TET1 is highly expressed in some cases of the hematological malignancies including acute myeloid leukemia. Indeed, studies by mouse models using conventional Tet1 knockout mice demonstrated that Tet1 is involved in myeloid leukemogenesis by Mixed Lineage Leukemia (MLL) fusion gene or TET2 mutant. Meanwhile, the other study showed that Tet1 is highly expressed in hematopoietic stem cells (HSCs), and that deletion of Tet1 in HSCs enhances potential self-renewal capacity, which is potentially associated with myeloid leukemogenesis. To examine the role of Tet1 in myeloid leukemogenesis more precisely, we generated novel conditional Tet1-knockout mice, which were used to generate the compound mutant mice by crossing with the inducible MLL-ENL transgenic mice that we developed previously. The leukemic immortalization in vitro was not critically affected by conditional ablation of Tet1 in HSCs with the induced expression of MLL-ENL or in hematopoietic progenitor cells retrovirally transduced with MLL-ENL. In addition, the leukemic phenotypes caused by the induced expression of MLL-ENL in vivo was not also critically affected in the compound mutant mouse model by conditional ablation of Tet1, although we found that the expression of Evi1, which is one of critical target genes of MLL fusion gene, in tumor cells was remarkably low under Tet1-ablated condition. These results revealed that Tet1 was dispensable for the myeloid leukemogenesis by MLL-ENL, suggesting that the therapeutic application of Tet1 inhibition may need careful assessment.

In-vitro modeling of TKI resistance in the high-risk B-cell acute lymphoblastic leukemia fusion gene RANBP2-ABL1 - implications for targeted therapy

Acute lymphoblastic leukemia remains a leading cause of cancer-related death in children. Furthermore, subtypes such as Ph-like ALL remain at high-risk of relapse, and treatment resistance remains a significant clinical issue. The patient-derived Ph-like ALL RANBP2-ABL1 fusion gene was transduced into Ba/F3 cells and allowed to become resistant to the tyrosine kinase inhibitors (TKIs) imatinib or dasatinib, followed by secondary resistance to ponatinib. RANBP2-ABL1 Ba/F3 cells developed the clinically relevant ABL1 p.T315I mutation and upon secondary resistance to ponatinib, developed compound mutations, including a novel ABL1 p.L302H mutation. Significantly, compound mutations were targetable with a combination of asciminib and ponatinib. In-vitro modeling of Ph-like ALL RANBP2-ABL1 has identified kinase domain mutations in response to TKI treatment, that may have important clinical ramifications. Early detection of mutations is paramount to guide treatment strategies and improve survival in this high-risk group of patients.

Prognostic significance of SET-NUP214 fusion gene in acute leukemia after allogeneic hematopoietic stem cell transplantation.

The SET nuclear proto-oncogene (SET)-nucleoporin (NUP) 214 fusion gene (SET-NUP214) is a rare leukemia fusion gene. Due to the limited number of samples with SET-NUP214 fusion gene in previous studies, the significance of SET-NUP214 for measurable residual disease (MRD) monitoring in patients with acute leukemia (AL) is still unclear. Our study aimed to observe the dynamic changes in SET-NUP214 expression before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and analyzed whether SET-NUP214 could be used to evaluate MRD status. Our study included 24 AL patients who were newly diagnosed with SET-NUP214 fusion gene and they all received allo-HSCT. Their MRD was evaluated by monitoring SET-NUP214 fusion gene and leukemia-associated immunophenotype (LAIP). The median follow-up time was 501 days (56–2208 days). Of the enrolled patients, 6 (25%) patients died, including 3 (12.5%) patients died of leukemia relapse. Total 5 (20.8%) patients experienced hematological relapse at a median of 225 days (56–1057 days) post-transplantation. The SET-NUP214 median expression level at diagnosis was 405.1% (14.6%–1482.4%). SET-NUP214 gene expression generally became positive prior to flow cytometry results. In addition, the Kaplan-Meier survival curves analysis showed that those who had SET-NUP214 positive (SET-NUP214+) post-transplantation had a higher 2-year cumulative incidence of leukemia relapse (CIR) of 43.7 ± 18.8% (P < .05). However, there was no significant difference between SET-NUP214 positive and SET-NUP214 negative patients with regard to their 2-year overall survival (OS) (82.5 ± 11.3 vs 64.6 ± 17.5%, respectively, P = .271). ROC curve analysis turned out that the area under the ROC curve (AUC) was 0.916 (95% CI: 0.784–1.0; P = .005). In conclusion, SET-NUP214 fusion gene determined by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) could be used to evaluate MRD status after allo-HSCT. Patients with positive SET-NUP214 expression after transplantation will have a poor prognosis.

Core Binding Factor Leukemias Utilize a Physiologic Sense/Antisense Promoter Switch Employed By T-Cells

Introduction: Alterations of core binding factors (CBF), Runx1 and CBFβ are frequent mutational targets in acute myeloid leukemia (AML). Chromosomal translocations t(8;21)(q22;q22) and inv(16)(p13q22), creating the fusion proteins RUNX1-ETO and CBFβ-MYH11 respectively, account for 15% and thus the largest sub-group of AML called CBF-AML. CBF oncogenes induce global changes in chromatin structure and gene regulation, which lead to differentiation blockade. A critical leukemic event could be the inactivation of PU.1 transcription factor. Normal myeloid differentiation needs PU.1 levels to increase, failure to do so leads to a stop of differentiation and AML development. In contrast, T-cell differentiation requires PU.1 to be completely switched off. The exact mechanism of PU.1 suppression, physiological for T-lymphopoiesis or pathological for leukemia, remains elusive. Results: We assessed the activation of the PU.1 locus throughout human hematopoietic differentiation stages using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Interestingly, we observed high accessibility of a previously identified antisense promoter (AsPr) in intron 3 and antisense transcript (asRNA) expression during early lymphopoiesis which preceded locus shutdown in T cells. The ratio of AsPr/PrPr accessibility and of antisense/sense transcription clearly indicated cellular fate during hematopoiesis (Figure 1A). T-lymphoid differentiation was related to the timely expression of RUNX transcription factors. RUNX1, RUNX3, and the CBF fusions RUNX1-ETO and CBFβ-MYH11 were capable to transactivate PU.1 AsPr. In CBF-AML patient samples we strikingly found elevated asRNA/mRNA ratios compared to normal karyotype AML or healthy CD34+ cells (Figure 1B) and increased AsPr/PrPr ratios unsing DNaseI-seq data in RUNX1-ETO AML patients. Functionally we found that PU.1 asRNA depletion in t(8;21) xenografted immune-deficient (NOD/SCID) mice restored a normal survival (Figure 1C) demonstrating that PU.1 antisense transcripts are required for CBF leukemia outgrow in vivo. To further dissect the mechanism of how CBFs could drive PU.1 antisense transcription we applied active RNA polymerase mapping (PRO-seq) and chromatin accessibility (ATAC-seq) and found a shift from PU.1 antisense to sense transcription after RUNX1-ETO depletion. Using chromosomal conformation capture sequencing (3C, Hi-C and CHiC) in T-lymphoid, myeloid and RUNX1-ETO cells combined with transcript quantification we observed that competitive interaction of an upstream enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development (Figure 2A). Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription (Figure 2B). Conclusion: The data suggest that silencing transcription factor PU.1 is an active process that requires a specific chromosome formation that is induced by CBF fusions. Sense/antisense promoter competition represents a crucial functional switch for gene expression perturbation by oncogenes and provide a potential strategy for future precise therapeutic targeting of oncogene-induced chromatin remodeling. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Staber:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; msd: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria.

Activation of CpG-Rich Promoters Mediated by MLL Drives MOZ-Rearranged Leukemia

Uncontrolled self-renewal of hematopoietic progenitors induces leukemia. To self-renew, leukemia cells must continuously activate genes that were previously active in their mother cells. Here, we describe the circuitry of a transactivation system responsible for oncogenic self-renewal. MLL recruits RNA polymerase II (RNAP2) to unmethylated CpG-rich promoters by its CXXC domain and activates transcription by transcriptional regulators, including the AF4 family/ENL family/P-TEFb complex, DOT1L, and p300/CBP histone acetyl transferases. MOZ also targets a broad range of CpG-rich promoters through association with RNAP2 and MLL. Leukemic fusion proteins such as MOZ-TIF2 and MLL-AFX constitutively activate CpG-rich promoters by aberrantly recruiting p300/CBP. Pharmacological inhibition of MLL or DOT1L induces differentiation of MOZ-TIF2-transformed cells. These results reveal that activation of unmethylated CpG-rich promoters mediated by MLL is the central mechanism of oncogenic self-renewal in MOZ-rearranged leukemia and indicate that the molecularly targeted therapies intended for MLL-rearranged leukemia can be applied for MOZ-rearranged leukemia.

Clinical and molecular consequences of fusion genes in myeloid malignancies

Leukemias are heterogeneous diseases characterized by aberrant hematopoietic stem and progenitor cells (HSPCs). Oncogenic fusion genes and proteins, produced via gross chromosomal rearrangements, such as chromosomal translocation, insertion, and inversion, play important roles in hematologic malignancies. These oncoproteins alter fundamental cellular properties, such as self-renewal, differentiation, and proliferation, and confer leukemogenic potential to HSPCs. In addition to providing fundamental insights into the process of leukemic transformation, these fusion genes provide targets for treatment and monitoring of myeloid leukemias. Furthermore, new technologies such as next-generation sequencing have allowed additional insights into the nature of leukemic fusion genes. In this review, we discuss the history, biologic effect, and clinical impact of fusion genes in the field of myeloid leukemias.

Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.

Exploitable metabolic dependencies in MLL-ENL–induced leukemia

AbstractMixed-lineage leukemia (MLL) fusions are transcriptional activators that induce leukemia, with a dismal prognosis that mandates further elucidation of their transformation mechanism. In this study, knockdown of the direct MLL-ENL target gene polypyrimidine tract binding protein-1 (PTBP1) was rate limiting for cell proliferation and caused a metabolic phenotype associated with reduced glucose consumption and lactate production. This effect was accompanied by a reduction of splice isoform-2 of pyruvate kinase M (PKM2). Because PKM2 restricts glycolytic outflow to provide anabolic intermediates, we tested the consequences of glucose, energy, and Ser/Gly starvation for cell physiology. Administration of deoxyglucose, energetic decoupling with rotenone, and inhibition of Ser biosynthesis by CBR5884 had a significantly stronger influence on self-renewal and survival of transformed cells than on normal controls. In particular, inhibition of Ser synthesis, which branches off glycolysis caused accumulation of reactive oxygen species, DNA damage, and apoptosis, predominantly in leukemic cells. Depletion of exogenous Ser/Gly affected proliferation and self-renewal of murine and human leukemia samples, even though they are classified as nonessential amino acids. Response to Ser/Gly starvation correlated with glucose transport, but did not involve activation of the AMPK energy homeostasis system. Finally, survival times in transplantation experiments were significantly extended by feeding recipients a Ser/Gly-free diet. These results suggest selective starvation as an option for supportive leukemia treatment.