Leukemic Progenitor Research Articles

CD200 Is a Stem Cell-Specific Immunosuppressive Target in AML

Acute myeloid leukemia (AML) stem cells (LSC) are an extremely rare fraction of the overall disease (likely <0.3%), largely quiescent, and capable of both long-term self-renewal and production of more differentiated leukemic blasts. Besides their role in disease initiation, they are also hypothesized as the likely source of deadly, relapsed leukemia. Due to the quiescent nature of the LSCs, they are capable of evading the majority of chemotherapeutic agents that rely on active cell-cycling for cytotoxicity. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML.We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression data to identify genes significantly over-expressed in LSCs when compared to their normal hematopoietic stem cell (HSC) counterparts (Herbrich et al Blood 2017 130:3962). These datasets contain gene expression arrays on human AML patient samples sorted by leukemia stem, progenitor, and blast cells (with normal hematopoietic cell subsets for comparison). We have since expanded our statistical model to identify targets that are both significantly overexpressed in AML LSCs when compared to HSC as well as LSCs compared to their corresponding, more differentiated blast cells. Instead of traditional methods for multiple testing corrections, we looked at the intersection of genes that met the above criteria in 3 independently generated datasets. This resulted in a list of 30 genes, 28 of which appear to be novel markers of AML LSCs.From this list, we first chose to focus on CD200, a type-1 transmembrane glycoprotein. CD200 is broadly expressed on myeloid, lymphoid, and epithelial cells, while the CD200 receptor (CD200R) expression is strictly confined to myeloid and a subset of T cells. CD200 has been shown to have an immunosuppressive effect on macrophages and NK cells and correlates with a high prevalence FOXP3+ regulatory T cells (Coles et al Leukemia 2012; 26:2146-2148). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget 2015; 6:30212-30221). To date, we have screened 20 primary AML patient samples by flow cytometry, 90% of which are positive for CD200. Expression is significantly enriched in the CD34+/CD123+ stem cell compartment.To examine the role of CD200 in AML, we established two in vitro model systems. First, we used CRISPR/Cas9 to knockout the endogenous CD200 protein in Kasumi-1. Further, we induced CD200 in the OCI-AML3 cell line that had no expression at baseline. Both cell lines did not express the CD200 receptor before or after manipulation, negating any autocrine signaling. In both systems, CD200 manipulation did not affect the proliferation rate or viability of the cells.To examine the immune function of CD200 in AML, we performed a series of mixed lymphocyte reactions. We cultured normal human peripheral blood mononuclear cells (PBMCs) with the CD200+ or CD200- cells from each line both. Cells were incubated in the culture media for 4-48 hours before being harvested and measured by flow cytometry for apoptosis or intracellular cytokine production. The presence of CD200 on the cell surface reduced the rate of immune-specific apoptosis among these leukemia cells. The difference in cell killing was most likely attributable to a CD200-specific suppression of CD107a, a surrogate marker or cytotoxic activity. In the OCI-AML3 model, PBMCs co-cultured with CD200+ cells produced approximately 40% less CD107a when compared to the CD200- co-culture. Additionally, we characterized our new cell lines using RNA sequencing. By comparing the CD200+ to the CD200- cells within each line, we observed that CD200+ cells significantly downregulate genes involved in defining an inflammatory response as well as genes regulated by NF-κB in response to TNFα. This indicates that CD200 may have an undiscovered intrinsic role in suppressing the immune microenvironment of AML LSCs.In conclusion, we have expanded our novel bioinformatics approach for robustly identifying AML LSC-specific targets. Additionally, we have shown that one of these markers, CD200, has a potential role as a stem cell-specific immunosuppressive target by reducing immune-mediated apoptosis and transcriptionally suppressing inflammatory cell processes. We are extending our study to explore CD200 in primary patient samples using a CD200-blocking antibody. DisclosuresAndreeff:SentiBio: Equity Ownership; Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Reata: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Astra Zeneca: Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

Characterizing Population Heterogeneity and Signaling Changes in Chronic Myeloid Leukemia Stem and Progenitor Cells upon Combined Treatment with Imatinib and MEK Inhibitors Using Quantitative Single Cell Phospho-Imaging

Chronic myeloid leukemia (CML) is caused by the oncogenic tyrosine kinase BCR-ABL1, which is uniquely present in the leukemic cells and drives progression of the disease. Small-molecule ABL1 tyrosine kinase inhibitors (TKIs) have transformed prognosis for patients as compared to interferon or standard chemotherapy, with the vast majority of patients achieving deep, long-term remission on treatment (Druker et al., Nature Medicine 2009). However, results from multiple TKI discontinuation studies over the past decade suggest approximately 50-60% of patients must remain on therapy indefinitely, highlighting challenges associated with the retention of a residual pool of BCR-ABL1-positive leukemic stem cells (LSCs) (Mahon et al., The Lancet 2018; Ross et al., Blood 2013). Previously we have shown that, while ABL1 TKIs can effectively inhibit BCR-ABL1 kinase activity and reduce CML disease burden, LSCs avoid ABL1 TKI-induced cell death via alternate signaling pathways such as MEK/ERK and are able to persist (Corbin et al., JCI 2011). Detecting changes in phospho-activation of such alternative pathways at the single cell level using primary CML samples has yet to be characterized and will inform therapeutic options for complete disease eradication. Here, we dissect phospho-signaling diversity with single cell level granularity across heterogeneous subpopulations of CML stem (CD34+CD38-) and progenitor (CD34+CD38+) cells upon drug treatment using a new quantitative imaging method that allows for sensitive detection of critical changes in signaling (Jacob et al., Scientific Reports 2016). Briefly, primary CD34+ cells were isolated from newly diagnosed TKI naïve CML patients by MACS column and cultured ex-vivo in the presence of imatinib and MEK inhibitors (trametinib or cobimetinib), either alone or in combination. Cells were then assessed for apoptosis by flow cytometry annexin assay, followed by fixing/staining for CD38, pCRKL, and pERK, and quantified for changes in signaling upon treatment by quantitative imaging method. This new phospho-imaging method allows for low cell number input and maximizes data output per sample, opening opportunities to assess translational applicability of combinations in a highly biologically relevant context of small but critical LSC populations. Quantification of phospho-signal revealed that pCRKL was reduced in a dose dependent manner across a range of concentrations of imatinib in both CML stem and progenitor cells. In contrast, while pERK levels were also reduced with imatinib treatment, a subset of CML stem cells exhibited high levels of pERK signaling even at high (5uM) concentrations of imatinib. Combination treatment with imatinib and trametinib or cobimetinib markedly increased apoptosis in CML CD34+ cells compared to each single agent. Within the stem cell population, median pERK fluorescence was significantly reduced with imatinib or trametinib compared to untreated control (p<0.0001), and further reduced with the combination compared to each single agent (p<0.0001 and p=0.04, respectively). Furthermore, the percent of cells with pERK levels below the untreated median value was 87%, 90% and 97% upon treatment with imatinib, trametinib or the combination respectively. Together, our data suggest that variable levels of ERK phosphorylation exist within the CML stem and progenitor compartments, including a subset of CML stem cells that exhibit persistently high levels of pERK despite effective suppression of BCR-ABL1 kinase activity by imatinib. This stem cell persistence warrants the need for combination strategies to inhibit BCR-ABL1 and MEK/ERK for their complete elimination. DisclosuresTyner:AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Leap Oncology: Equity Ownership; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Agios: Research Funding. Druker:Celgene: Consultancy; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oregon Health & Science University: Patents & Royalties; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Novartis Pharmaceuticals: Research Funding; ARIAD: Research Funding; Millipore: Patents & Royalties; Amgen: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding.

Results of a Phase 2, Open-Label Study of Idarubicin (I), Cytarabine (A) and Nivolumab (Nivo) in Patients with Newly Diagnosed Acute Myeloid Leukemia (AML) and High-Risk Myelodysplastic Syndrome (MDS)

Results of a Phase 2, Open-Label Study of Idarubicin (I), Cytarabine (A) and Nivolumab (Nivo) in Patients with Newly Diagnosed Acute Myeloid Leukemia (AML) and High-Risk Myelodysplastic Syndrome (MDS)

Dwjm-Adherence Induces Chemotherapy Resistance in Primary Acute Myeloid Leukemia By Altering Leukemia Cell Metabolism

Introduction: In previous work, we have demonstrated that culturing leukemia cell lines in decellularized Wharton's jelly (WJ) matrix (DWJM), the gelatinous material in umbilical cord tissues, resulted in chemotherapy resistance. To reduce the variability in the biochemical composition between different parts of the WJ matrix and to optimize our 3-dimensional (3D) DWJM-based extracellular matrix (ECM) model for in vitro culture of primary AML cells, we fabricated DWJM-derived porous disks with uniform architecture and pore-size by homogenization followed by lyophilization of human DWJM. Herein, we examine whether DWJM disks support primary leukemia cells and result in chemotherapy resistance.Methods: AML patient samples collected by leukapheresis were cultured in DWJM disks for one week. Non-adherent cells were first aspirated and adherent cells were separately isolated and assessed for viability, apoptosis, and colony forming unit (CFU). RNA sequencing was performed at the end of culture. Response to chemotherapy following treatment with doxorubicin was also assessed. For all studies, DWJM-adherent and non-adherent cells were compared to suspension culture controls. Metabolic pathway analyses were conducted using enzyme-linked immunosorbent assay (ELISA). One-tailed t-test was used for comparison between the groups.Results: Consistent with our prior studies, adherent DWJM cells demonstrated less apoptosis (P=0.027) and greater CFU activity with larger and more dense colonies (1 vs 4/50,000 cells, P=0.0008). Co-culture of primary AML samples with DWJM reduced doxorubicin induced cell death (P=.047) preserving CFU activity (3.3 vs 0.3/300,000 cells, P=.047) compared to treatment of suspension culture cells. To understand the mechanisms by which co-culture with DWJM enhanced leukemic progenitor function and therapy resistance, we performed RNA-Seq analyses. RNA-Seq data analysis from day 7 demonstrated significant upregulation of FAM83A and MIR34A and downregulation of BPI, ZNF521, NHLH2, CD69, FKBP14, PBX1, TANC1, GRIN2b, MYO6, INHBA, SA1008, CXCL1, A1009, BLNK, MMP9, BHLHE41, and CD9 in the adherent population compared to the suspension population (adjusted P-value <0.05). Ingenuity Pathway Analysis (IPA) identified glutamate receptor signaling as the top impacted canonical pathway, suggesting differences in the predominant metabolic process between the two culture conditions. Additionally, IPA showed that FAM83A was the most upregulated molecule, while MT-TH and MT-TW were two of the most down-regulated molecules in the adherent cells. The DWJM culture condition was associated with a significant increase in lactate (P=0.020) and significant reduction in glucose (P=0.005) in culture supernatants compared to suspension controls.Conclusion: We demonstrate that DWJM disks support primary leukemia cell survival. DWJM-adherent cells demonstrate chemotherapy resistance in association with induction of glycolysis. Based on our RNA Seq data we hypothesize that leukemia cell adherence to DWJM upregulates FAM83A, which functions in the epidermal growth factor receptor (EGFR) signaling pathway. FAM83A is known to control PI3K-AKT-TOR signaling cascade. M-TOR, in turn is known to activate HIF-1α pathway, which regulates glycolysis (Figure-1). Additionally, down-regulation of mitochondria-related molecules (MT-TH and MT-TW) is consistent with a switch from oxidative phosphorylation to glycolysis in the adherent cells. Further work is ongoing to further understand the role of glycolysis in primary leukemia cell chemotherapy resistance. [Display omitted] DisclosuresLipe:Celgene: Consultancy. Aljitawi:The University of Rochester Medical Center: Patents & Royalties: Pending patent related to decellularized Wharton's jelly matrix; Medpace: Consultancy.

Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia.

The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the −11.7 kb and −0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34− cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.

RACK1 deficiency synergizes with all-trans retinoic acid to induce apoptosis in human acute promyelocytic leukemia cells.

As a classic differentiation agent, all-trans retinoic acid (ATRA) has been widely used in the treatment of acute promyelocytic leukemia (APL). However, the clinical application of ATRA has strict limitations, for its severe side effects due to the accumulation of peripheral blood leukocytes. The scaffold protein RACK1 (Receptor for activated C kinase 1), which regulates multiple signaling pathways, has been proposed to contribute to the survival of leukemic progenitors. But it remains unclear whether it is also involved in the oncogenic growth of APL. In the present study, we demonstrate that silencing of endogenous RACK1 expression synergized with ATRA to promote the death of NB4 and HL-60 APL cells without effect on cell differentiation induced by ATRA. Interestingly, RACK1 knockdown combined with ATRA treatment mainly induces apoptosis. It is distinct to the necrotic cell death induced by idarubicin in combination with ATRA, a regimen extensively used in the clinic to prevent neutrophil accumulation. Further exploration revealed that the lysosome-autophagy pathway is likely to be responsible for the anti-apoptotic role of RACK1. Taken together, our findings indicate that RACK1 is essential in maintaining the malignant features of APL, and targeting RACK1 may have promising therapeutic implications in the treatment of APL.

Tyrosine kinase inhibitor–induced defects in DNA repair sensitize FLT3(ITD)-positive leukemia cells to PARP1 inhibitors

AbstractMutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.

CD16+NK-92 and anti-CD123 monoclonal antibody prolongs survival in primary human acute myeloid leukemia xenografted mice.

Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.

Abstract 1114: IGFBP7 reduces acute myeloid leukemia stem cell survival without affecting normal hematopoiesis

Abstract Only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. This poor prognosis is mainly caused by treatment failure due to chemotherapy resistance and relapse. Leukemic stem cells (LSCs) are thought to be the major determinants of AML relapse due their potential for self-renewal and chemotherapy resistance. It has been demonstrated that LSC frequency and expression of LSC gene expression signatures is highly predictive of therapy failure in AML patients, indicating the clinical importance of LSCs. LSCs co-reside with residual normal hematopoietic stem cells (HSCs) in the diseased bone marrow. Increasing the dose of chemotherapy might eliminate LSCs, however will inevitable result in non-specific elimination of HSCs, delaying or preventing recovery of normal hematopoiesis after treatment. To significantly improve AML patients outcome, there is an urgent need for identification of alternative therapies that specifically eliminate LSCs while sparing HSC. We generated gene expression profiles of HSCs, LSCs and leukemic progenitors all derived from the same AML bone marrow and identified insulin growth factor binding protein 7 (IGFBP7) as one of the top differentially expressed genes. We found that low IGFBP7 is a feature of LSCs, associated with reduced chemotherapy sensitivity and that IGFBP7 is frequently downregulated at relapse as compared to AML diagnosis. Together, these results suggest that there is a survival advantage of IGFBP7low AML cells during chemotherapy treatment. To test whether increasing IGFBP7 levels might be a strategy to deplete leukemic (stem) cells, we overexpressed IGFBP7 or added recombinant human IGFBP7 (rhIGFBP7) to primary AML cells. Strikingly, IGFBP7 overexpression or addition of rhIGFBP7 led to induction of differentiation and cell death in AML patient cells. Moreover, rhIGFBP7 reduces leukemic engraftment in an AML xenograft mouse model, reverses a stem-like gene signature, and inhibits AML blast and leukemic stem/progenitor cell survival in vitro and in vivo, while it had no influence on normal hematopoietic (stem) cell survival. Our findings define an association between chemotherapy sensitivity and IGFBP7 expression levels in primary AML cells, and indicate that treatment of AML patients with a combination of rhIGFBP7 and chemotherapy might reduce AML residual disease and LSC survival. Citation Format: Han J. Verhagen, Noortje van Gils, Anna van Rhenen, Arjo Rutten, Marjon Smit, Mei-Ling Tsui, Louise L. de Vos Klootwijk, Renee X. Menezes, Meyram Cil, Margaretha G. Roemer, Fabio Brocco, Fedor Denkers, Eline Vermue, Stan Heukelom, Sonja Zweegman, Jeroen J. Janssen, Gert J. Ossenkoppele, Gerrit Jan Schuurhuis, Linda Smit. IGFBP7 reduces acute myeloid leukemia stem cell survival without affecting normal hematopoiesis [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 1114.

Abstract 935: Generation and preclinical characterization of CD123-CPI antibody-drug conjugate (ADC)

Abstract CD123 is expressed on cancer cells in a variety of hematologic malignancies including acute myeloid leukemia (AML). CD123 is frequently expressed on leukemic blasts and leukemic stem cells (LSCs), a cell population associated with relapse in patients, but there is minimal or no expression on most normal hematopoietic cells and solid tissues. Thus, CD123 is a promising target for AML. We have generated an anti-CD123 ADC that carries cyclopropylpyrroloindoline (CPI) payload that crosslinks DNA. A drug loading of 2 molecules of CPI per molecule of antibody was achieved by site-specific conjugation using our transglutaminase methodology. Upon binding to the CD123 antigen, CD123-ADC is internalized and delivered to the endosomal-lysosomal pathway whereupon the CPI payload is released from the antibody by proteolysis of the linker. The released CPI alkylates DNA, which activates ATR/ATM, CHK1, CHK2 and FANCD2, ultimately resulting in cell death. In vitro, CD123–CPI elicited cytotoxicity in a dose-dependent manner against several CD123-positive, but not against CD123-negative cell lines. Cell lines with higher CD123 expression level were more sensitive to the ADC. Long-Term Culture-Initiating Cells in vitro assay showed that AML patient bone marrow samples that naturally have high percentage of LSCs yielded substantially fewer colonies in CD123-ADC treated cells compared to control ADC. Importantly, CD123-ADC had no adverse effects in healthy donor bone marrow cells experimentally enriched in CD34+ primitive stem cells. This result suggests that CD123-ADC specifically inhibits the growth potential of leukemic blasts and progenitor cells. In vivo, robust antileukemic activity was observed in CD123-positive AML cell line-derived xenograft models. Low doses of CD123-ADC effectively regressed tumors whereas the tumor progressed in mice that received control ADC. Efficacy was also evaluated in disseminated AML PDX models (n≥7) established with patient samples of various cytogenetics and molecular abnormalities, and relapse/refractory. Flow cytometry analyses of leukemic load in peripheral blood and in bone marrow samples of mice showed that CD123-ADC was efficacious in reducing tumor burden even at the suboptimal doses. In summary, our data demonstrate that our CD123-ADC is highly active in a broad panel of primary AML samples. Currently, CD123-ADC in combination with other AML therapies is being tested in vivo. All these attributes of CD123-ADC make it an attractive agent to evaluate in clinical trials. Citation Format: Yoon-Chi Han, Fan Jiang, Nicole Piche-Nicholas, Madan Katragadda, Nadira Prashad, Manoj Charati, Wendy Hu, Mauricio Leal, Nathan Tumey, Andreas Maderna, Russell Dushin, Kenny Kim, LuAnna Lemon, Marc Damelin, HP Gerber, Lioudmila Tchistiakova, Frank Loganza, Chris O'Donnell, Puja Sapra. Generation and preclinical characterization of CD123-CPI antibody-drug conjugate (ADC) [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 935.

Abstract 4957: The novel imipridone ONC212 highly synergizes with the BCL-2 inhibitor ABT-199 in AML and activates orphan receptor GPR132

Abstract Imipridones are first-in-class anti-tumor compounds including ONC201, which has shown promising clinical activity. ONC212 was designed as a second-generation imipridone. We first confirmed the ONC212 effects in a collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project; leukemia was identified as the most sensitive tumor type. In fact, ONC212 exerted prominent apoptogenic effects in acute myeloid leukemia (AML) cell lines and primary AML, but not normal bone marrow (BM) cells. We investigated the effects of ONC212 in vivo in an aggressive systemic AML xenograft model using OCI-AML3 cells. ONC212 markedly inhibited AML expansion and prolonged median survival (controls: 43 d, ONC212: 49 d; p = 0.0003). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells (LSPCs), we treated patient-derived xenograft (PDX) cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected into recipient NSG mice. After one month, the human leukemic CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212 treated group. The median survival was remarkably prolonged (controls: 36 d, ONC212: 82 d; p &amp;lt; 0.0001). These results indicate that ONC212 has anti-LSPC effects to reduce the engraftment potential. We previously demonstrated that the prototype it compound ONC201 induces apoptosis via an atypical integrated stress response (ISR; Ishizawa et al., Sci Signal, 2016). As expected, ONC212 induced the transcription factor ATF4, a key effector of ISR. Because BCL-2 is generally considered to be protective against ISR-mediated apoptosis, we hypothesized that the BCL-2 inhibitor ABT-199 could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 synergistically induced apoptosis in AML cells. Furthermore, the combination showed highly significant synergistic anti-leukemia effects in vivo. The combinatorial treatment prolonged overall median survival (controls: 20 d, each agent: 21 d, the combination 30 d; p &amp;lt; 0.0001). Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of ONC201, we hypothesized that ONC212 also targets GPCRs. The PathHunter β-arrestin screening discovered that ONC212 specifically activated the orphan GPCR GPR132. Consistently, the GPR132 mRNA expression was correlated with ONC212 sensitivity. On the other hand, GPR132 overexpression induced cell death in AML cells, which is consistent with previous reports implicating GPR132 as a tumor suppressor. Furthermore, ONC212 increased GPR132 mRNA expression. These results suggest that GPR132 could be a potential therapeutic target in AML. Taken together, ONC212 has potential as a novel agent for AML therapy. This study provides the first reported opportunity to therapeutically target GPR132 in oncology. Citation Format: Takenobu Nii, Jo Ishizawa, Varun V. Prabhu, Vivian Ruvolo, Neel Madhukar, Ran Zhao, Hong Mu, Lauren Heese, Kensuke Kojima, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Olivier Elemento, Joshua Allen, Wolfgang Oster, Martin Stogniew, Michael Andreeff. The novel imipridone ONC212 highly synergizes with the BCL-2 inhibitor ABT-199 in AML and activates orphan receptor GPR132 [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 4957.

Abstract 1134: The tumor suppressor activity of miR-300 is detrimental for leukemia development but required for leukemia stem cell maintenance

Abstract Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance and disease development. Persistence of drug-resistant quiescent LSCs depends on cell-autonomous and bone marrow (BM) signals. Herein, we identified miR-300 as an miRNA inhibited in CD34+ CML progenitors and during blastic transformation (BC) through the BCR-ABL1-dependent inhibition of C/EBPβ-induced miR-300 transcription. In CML progenitors, ectopic mir-300 expression directly targets the PP2A inhibitory pathway (i.e., Jak2, hnRNPA1, SET) and other factors essential for LSC maintenance and disease progression (e.g., CCND1/2, b-catenin, Myc, Twist-1). In leukemic but not normal CD34+ cells, miR-300 acts as a potent tumor suppressor by inducing cell cycle exit and promoting apoptosis. Conversely, hypoxia-induced BCR-ABL1 inhibition and induction of C/EBPβ were found essential for increased miR-300 levels in quiescent LSCs, although mesenchymal stromal cells (MSC)-derived exosomal miR-300 also contributed to it. Moreover, low O2 levels and MSC-derived exosomes induced quiescence of CD34+ CML cells. Notably, expression of an anti-miR-300 in MSCs prevented exosome-induced CD34+ CML growth arrest. LSCs escaped miR-300-induced apoptosis through the autocrine/paracrine TGFβ1-induced expression of TUG1, a lncRNA acting as an miR-300 sponge. In fact, TUG1 or TGFβ1 inhibition decreased quiescent (CFSEMAX) LSC number. By contrast, miR-300 inhibition did not alter LSC survival/self-renewal, further supporting a role for TUG1 as an miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low levels of ectopic miR-300 induced growth arrest (decreased LTC-IC and CFC/replating activity) without affecting quiescent LSC number. By contrast, high doses of miR-300 but not scramble CpG-ODN impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced marked killing of quiescent LSCs and dividing progenitors, and impaired CML engraftment in NRG-SGM3 mice. Such effects were further enhanced when CPG-miR-300 and CPG-anti-TUG1 were combined. By contrast, high CpG-miR-300 levels did not affect normal CD34+ cell survival/self-renewal likely because of high TUG1 expression. Altogether our results indicate that while miR-300 loss is essential for survival/proliferation of leukemic progenitors, increased miR-300 levels are required for LSC maintenance. Thus, induction of TUG1 may occur to preserve LSC survival in the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through abnormal miR-300 induction. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias. This work is supported in part by NIH-NCI R01CA163800. Citation Format: Giovannino Silvestri, Lorenzo Stramucci, Justin Ellis, Jason Harb, Paolo Neviani, Bin Zhang, Klara Srutova, Gabriel Pineda, Catriona Jamieson, Bruno Calabretta, Fabio Stagno, Paolo Vigneri, Georgios Nteliopoulos, Philippa May, Alistar Reid, Ramiro Garzon, Denis-Claude Roy, Martin Guimond, Peter Hokland, Michael Deininger, Garrett Fitzgerald, Chris Harman, Francesco Dazzi, Dragana Milojkovic, Jane Apperley, Guido Marcucci, Jianfei Qi, Katerina Machova-Polakova, Xiaoxuan Fan, Maria Baer, Rossana Trotta, Danilo Perrotti. The tumor suppressor activity of miR-300 is detrimental for leukemia development but required for leukemia stem cell maintenance [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 1134.

Pilot Study on Mass Spectrometry-Based Analysis of the Proteome of CD34⁺CD123⁺ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia.

Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrometry methods to analyze the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy, granulocyte-colony stimulating factor (G-CSF) mobilized stem cell donors were analyzed. In this Tandem Mass Tag (TMT) 10-plex labelling–based approach, 2070 proteins were identified with 171 proteins differentially abundant in one or both cellular compartments. This proof-of-principle-study demonstrates the potential of mass spectrometry to detect differentially expressed proteins in two compartment fractions of the entire proteome of leukemic stem cells, compared to their non-malignant counterparts. This may contribute to future immunotherapeutic target discoveries and individualized AML patient characterization.

Interaction between the immune system and acute myeloid leukemia: A model incorporating promotion of regulatory T cell expansion by leukemic cells

Population dynamics of regulatory T cells (Treg) are crucial for the underlying interplay between leukemic and immune cells in progression of acute myeloid leukemia (AML). The goal of this work is to elucidate the dynamics of a model that includes Treg, which can be qualitatively assessed by accumulating clinical findings on the impact of activated immune cell infusion after selective Treg depletion. We constructed an ordinary differential equation model to describe the dynamics of three components in AML: leukemic blast cells, mature regulatory T cells (Treg), and mature effective T cells (Teff), including cytotoxic T lymphocytes. The model includes promotion of Treg expansion by leukemic blast cells, leukemic stem cell and progenitor cell targeting by Teff, and Treg-mediated Teff suppression, and exhibits two coexisting, stable steady states, corresponding to high leukemic cell load at diagnosis or relapse, and to long-term complete remission. Our model is capable of explaining the clinical findings that the survival of patients with AML after allogeneic stem cell transplantation is influenced by the duration of complete remission, and that cut-off minimal residual disease thresholds associated with a 100% relapse rate are identified in AML.

Proliferation dynamics of acute myeloid leukaemia and haematopoietic progenitors competing for bone marrow space

Leukaemia progressively invades bone marrow (BM), outcompeting healthy haematopoiesis by mechanisms that are not fully understood. Combining cell number measurements with a short-timescale dual pulse labelling method, we simultaneously determine the proliferation dynamics of primitive haematopoietic compartments and acute myeloid leukaemia (AML). We observe an unchanging proportion of AML cells entering S phase per hour throughout disease progression, with substantial BM egress at high levels of infiltration. For healthy haematopoiesis, we find haematopoietic stem cells (HSCs) make a significant contribution to cell production, but we phenotypically identify a quiescent subpopulation with enhanced engraftment ability. During AML progression, we observe that multipotent progenitors maintain a constant proportion entering S phase per hour, despite a dramatic decrease in the overall population size. Primitive populations are lost from BM with kinetics that are consistent with ousting irrespective of cell cycle state, with the exception of the quiescent HSC subpopulation, which is more resistant to elimination.

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling

The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.

Design and synthesis of novel dasatinib derivatives as inhibitors of leukemia stem cells

We used the concept of bioisosteres to design and synthesize a novel series of dasatinib derivatives for the treatment of leukemia. Unfortunately, most of the dasatinib derivatives did not show appreciable inhibition against leukemia cell lines K562 and HL60. However, acrylamide compound 2c had comparable inhibitory activity with dasatinib against K562 cells (IC50 = 0.039 nM vs. 0.069 nM). And amide compound 2a and acrylamide compound 2c also had comparable inhibitory activity with dasatinib against the leukemia cell line HL60 (IC50 = 0.25 nM and 0.26 nM vs. 0.11 nM). Against the leukemia progenitor cell line KG1a, triazole compounds 15a and 15d–15f and oxadiazole compounds 24a–24d were more potent than dasatinib. In particular, the hydroxyl compounds 15a and 24a were about 64 and 180 fold more potent than dasatinib against KG1a cells (IC50 = 0.14 μM and 0.05 μM vs. 8.98 μM). Compounds 15a and 24a also inhibited colony formation in MCF-7 cells and inhibited cell migration in the cell wound scratch assay in B16BL6 cells. Moreover, hydroxyl compounds 15a and 24a had low toxicity in vivo.

To the Nuclear Region Occupied by Nucleolar Bodies in Human Leukaemic Myeloblasts of Kasumi 1 and K 562 Lineages

Previous observation demonstrated that measured nucleolar and nuclear diameters and the resulting calculated ratio might facilitate estimation of the approximate size of the nuclear region occupied by the nucleolar bodies. The size of nuclear regions occupied by nucleolar bodies decreased during the differentiation and maturation of leukaemic lymphocytes, but was constant for each differentiation or maturation stage. The present study was undertaken to provide more information on the approximate size of the nuclear regions occupied by nucleolar bodies in leukaemic granulocytic progenitors. Myeloblasts of established Kasumi 1 and K 562 cell lineages originating from human myeloid leukaemias were convenient models for such study because they represented only one and early differentiation stage of granulocytic progenitors. According to the results, the maximal and mean nucleolar body : maximal and mean nuclear diameter ratios in myeloblasts without heavy nuclear alterations were stable and not markedly influenced by the anti-leukaemic treatment or aging. Thus, the roughly estimated size of nuclear regions occupied by nucleolar bodies in these cells appeared to be similar and stable regardless of aging or anti-leukaemic treatment. In contrast, the anti-leukaemic treatment or aging in such myeloblasts induced marked reduction of the nucleolar biosynthetic activity reflected by the decreased number of nucleolar fibrillar centres.

Biotransformation of 20(R)-panaxatriol by the fungus Aspergillus flavus Link AS 3.3950

Microbial transformation of 20(R)-panaxatriol by the fungus Aspergillus flavus Link AS 3.3950 was performed. Four new (1–4), along with two previously reported metabolites (5 and 6), were obtained. Their chemical structures were elucidated on the basis of extensive spectroscopic analyses. Furthermore, the inhibitory effects of those compounds on K562/ADR, Du-145, Hela, MCF-7 and HepG2 cell lines were evaluated by MTT assay. Among them, compound 15β-hydroxy-20(R)-panaxatriol (4) exhibited selective inhibitory effects on human leukaemic progenitor cells K562/ADR through arresting cell cycle, which was associated with obvious decrease of cyclin B1, cyclin D1 and cyclin-dependent kinase (CDK) 1/2/4/6 protein expression.