Acute Myeloid Leukemia CD34 Research Articles

Interleukin 4 induces apoptosis of acute myeloid leukemia cells in a Stat6-dependent manner

Cytokines provide signals that regulate immature normal and acute myeloid leukemia (AML) cells in the bone marrow microenvironment. We here identify interleukin 4 (IL4) as a selective inhibitor of AML cell growth and survival in a cytokine screen using fluorescently labeled AML cells. RNA-sequencing of the AML cells revealed an IL4-induced upregulation of Stat6 target genes and enrichment of apoptosis-related gene expression signatures. Consistent with these findings, we found that IL4 stimulation of AML cells induced Stat6 phosphorylation and that disruption of Stat6 using CRISPR/Cas9-genetic engineering rendered cells partially resistant to IL4-induced apoptosis. To evaluate whether IL4 inhibits AML cells in vivo, we expressed IL4 ectopically in AML cells transplanted into mice and also injected IL4 into leukemic mice; both strategies resulted in the suppression of the leukemia cell burden and increased survival. Notably, IL4 exposure caused reduced growth and survival of primary AML CD34+CD38− patient cells from several genetic subtypes of AML, whereas normal stem and progenitor cells were less affected. The IL4-induced apoptosis of AML cells was linked to Caspase-3 activation. Our results demonstrate that IL4 selectively induces apoptosis of AML cells in a Stat6-dependent manner—findings that may translate into new therapeutic opportunities in AML.

Cooperative effect of chidamide and chemotherapeutic drugs induce apoptosis by DNA damage accumulation and repair defects in acute myeloid leukemia stem and progenitor cells

BackgroundMany conventional chemotherapeutic drugs are known to be involved in DNA damage, thus ultimately leading to apoptosis of leukemic cells. However, they fail to completely eliminate leukemia stem cells (LSCs) due to their higher DNA repair capacity of cancer stem cells than that of bulk cancer cells, which becomes the root of drug resistance and leukemia recurrence. A new strategy to eliminate LSCs in acute myeloid leukemia (AML) is therefore urgently needed.ResultsWe report that a low-dose chidamide, a novel orally active benzamide-type histone deacetylase (HDAC) inhibitor, which selectively targets HDACs 1, 2, 3, and 10, could enhance the cytotoxicity of DNA-damaging agents (daunorubicin, idarubicin, and cytarabine) in CD34+CD38− KG1α cells, CD34+CD38− Kasumi cells, and primary refractory or relapsed AML CD34+ cells, reflected by the inhibition of cell proliferation, induction of apoptosis, and increase of cell cycle arrest in vitro. Mechanistically, these events were associated with DNA damage accumulation and repair defects. Co-treatment with chidamide and the DNA-damaging agent IDA gave rise to the production of γH2A.X and inhibited posttranslationally but not transcriptionally the repair gene of ATM, BRCA1, and checkpoint kinase 1 (CHK1) and 2 (CHK2) phosphorylation. Finally, the combination of chidamide and IDA initiated caspase-3 and PARP cleavage, but not caspase-8 and caspase-9, and ultimately induced CD34+CD38− KG1α cell apoptosis. Further analysis of AML patients’ clinical characteristics revealed that the ex vivo efficacy of chidamide in combination with IDA in primary CD34+ samples was significantly correlated to peripheral blood WBC counts at diagnosis, while LDH levels and karyotype status had no effect, indicating that the combination regimen of chidamide and IDA could rapidly diminish tumor burden in patients with R/R AML.ConclusionsThese findings provide preclinical evidence for low-dose chidamide in combination with chemotherapeutic agents in treating recurrent/resistant AML as an alternative salvage regimen, especially those possessing stem and progenitor cells.

Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34+ AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53mut), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53R273H. AML CD34+ cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34+ cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53wt), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53mut. To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34+ cells were separated into ROSlow and ROShigh subfractions. The immature AML CD34+-enriched ROSlow cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROShigh cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34+ cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction.

Abstract 4087: Development of a potent, dual pan-PIM/FLT3 inhibitor for the treatment of heme malignancies

Abstract Despite huge effort spent on understanding the pathogenesis of acute myeloid leukemia (AML), current standards of care are still based on the same chemotherapy agents as two decades ago - combinational treatment of cytarabine with an anthracycline. Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in AML. Although FLT3 inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. Selvita has developed a potent and selective first-in-class, dual PIM/FLT3 kinase inhibitor, the SEL24-B489 compound, and profiled its activity for in vitro and in vivo AML models showing significantly broader anti-tumor activity of SEL24-B489 than selective FLT3-ITD or PIM inhibitors. We compared SEL24-B489 head-to-head with a selective PIM inhibitor (AZD1208) and a selective FLT3-ITD inhibitor (AC220) in a panel of AML cell lines with FLT3-ITD or unmutated kinase (FLT3-WT) as well as peripheral AML cells and CD34+ bone marrow blasts. SEL24-B489 exhibited a significantly broader activity, irrespective of FLT3 status, than either of the selective inhibitors. Since PIM kinases have emerged as important mediators of FLT3-inhibitor resistance, we hypothesized that the dual specificity of SEL24-B489 might overcome the phenotype of resistance. We utilized previously developed MOLM-14 cells transduced with either FLT3-WT or FLT3 alleles containing TKD point mutations to show that neither of the these mutations decreased the cellular sensitivity to SEL24-B489. Higher cellular activity and biomarker response of SEL24-B489 than competitive inhibitors was shown by inhibition of specific biomarkers such as S6 and STAT5 phosphorylation at nanomolar concentrations in both FLT3-ITD positive and FLT3-WT cell lines in vitro. We have also demonstrated SEL24-B489 superior potency of SEL24-B489 in xenograft models in vivo. Consistent with the experiments in vitro showing marked synergy between SEL24-B489 and AraC, a combination of these agents resulted in almost completely blocked tumor growth in vivo. Most importantly, SEL24-B489 has been selected as a clinical candidate and is currently in phase I clinical trials. Citation Format: Wojciech Czardybon, Renata Windak, Aniela Gołas, Michał Gałęzowski, Aleksandra Sabiniarz, Izabela Dolata, Magdalena Salwińska, Paweł Guzik, Magdalena Zawadzka, Ewelina Gabor-Worwa, Bożena Winnik, Małgorzata Żurawska, Ewa Kolasińska, Ewelina Wincza, Marta Bugaj, Monika Danielewicz, Grzegorz Dubin, Ewa Jabłońska, Maciej Szydłowski, Tomasz Sewastianik, Bartosz Puła, Anna Szumera-Ciećkiewicz, Monika Prochorec-Sobieszek, Elżbieta Mądro, Ewa Lech-Marańda, Krzysztof Warzocha, Jerome Tamburini, Przemysław Juszczyński, Krzysztof Brzózka. Development of a potent, dual pan-PIM/FLT3 inhibitor for the treatment of heme malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4087. doi:10.1158/1538-7445.AM2017-4087

Blocking the Autophagy Pathway As Potential Target for the Treatment of Wild Type P53 AMLs

In normal hematopoiesis, autophagy is essential for stem and progenitor cell (HSPC) function. We previously reported that basal autophagy is higher in HSPCs compared to more differentiated cells, and genetic interference with the autophagy machinery abolished in vivo engraftment of cord blood (CB) CD34+ cells (Stem Cells 2016).To study the role of autophagy in acute myeloid leukemia (AML), steady-state autophagy was measured in a panel of myeloid leukemic cell lines (n=7) and primary human CD34+ AML cells (N=55). Autophagic flux was determined by the relative accumulation of cyto-ID (a marker for autophagic vesicles), measured by flow cytometry. Alternatively, autophagy was determined by LC3-II/p62 accumulation using Western blotting or fluorescence microscopy. Large variability in basal autophagy was observed in AML samples in contrast to CB CD34+ cells. Autophagic flux was significantly higher in AML CD34+ cells of patients belonging to poor-risk group (n=15) vs. intermediate and good-risk group (n=26, p=<0.001). Moreover, expression levels of the essential autophagy genes ATG5 and ATG7 were increased in AML CD34+ cells compared to normal bone marrow (NBM) CD34+ cells. Autophagy was functionally relevant for leukemic cells, because primary AML CD34+ cells (n=35) showed an increased susceptibility for the autophagy inhibitor hydroxychloroquine (HCQ), compared to NBM CD34+ cells (10µM HCQ, NBM CD34+: 84% ± 4 survival vs. AML CD34+: 43% ± 4, p=<0.001).The blocking of autophagy by genetic interference (lentiviral shATG5 or shATG7) or by a pharmacological approach (HCQ) triggered an apoptotic response in leukemic cells,which coincided with increased expression of p53 and the downstream pro-apoptotic target genes BAX and PUMA, the putative AKT inhibitor PHLDA3 and the cell cycle inhibitor p21. This apoptotic phenotype could be rescued by the lentiviral shRNA mediated knockdown of p53. Interestingly, AMLs harboring TP53 mutations (TP53mut, N=6) did not respond to HCQ treatment compared to wild type P53 (P53wt) AMLs. In line, the pro-apoptotic BAX and PUMA expression was not upregulated upon HCQ treatment, suggesting that the apoptotic response was severely dampened in TP53mut AMLs. Co-treatment with Nutlin-3 (which stabilizes p53), enhanced the cytoxic effect of HCQ in P53wt AMLs, but not TP53mut AMLs. In contrast, overexpression of mutant p53R273H in the p53wt cell lines OciM3 and Molm13, rendered cells more resistant to HCQ treatment.To further characterize autophagy in the leukemic stem cell (LSC) enriched cell fraction we separated AML CD34+ cellsin ROSlow and ROShigh cells, since previous studies have shown that LSCs are enriched in the ROSlow cell fraction. The CD34+ AML ROSlowcells had significantly lower mitochondrial mass and maintained a higher autophagy flux in comparison to the AML ROShigh cells (n=11, p=<0.05). In line, the autophagy regulator FOXO3A and the essential autophagy genes Beclin-1 and LC3 were expressed significantly higher in AML ROSlow cells. The increase in autophagy in AML ROSlowcells was also functionally relevant since these AML cells were more sensitive to HCQ treatment when compared with AML ROShigh cells. To validate the efficacy of autophagy inhibition in vivo, AML CD34+ cells of two different patients were transduced with the shATG5 or shSCR-GFP and subsequently transplanted in immunodeficient NSG mice. Inhibition of autophagy utilizing shATG5 prevented the in vivo engraftment of AML CD34+ cells in NSG mice.In conclusion, our results indicate that AML CD34+ cells are highly dependent on the autophagy machinery for growth and cell survival, in a p53-dependent manner. Targeting autophagy, potentially in conjunction with conventional chemotherapeutics might provide new therapeutic options for treatment of P53wt AMLs (Figure 1). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

Enhanced Targeting of FLT3-ITD+AML Stem Cells through Combined Inhibition of SIRT1 and Autophagic Flux

Internal tandem duplication mutations of the Fms-like tyrosine kinase 3 (FLT3-ITD) are amongst the most common mutations in acute myeloid leukemia (AML) patients, and are associated with poor clinical outcome. Tyrosine kinase inhibitors (TKI) targeting FLT3 are only partially effective and fail to yield full and lasting responses in FLT3-ITD+ AML patients. Therefore, additional strategies to enhance eradication of FLT3-ITD+ AML cells are required to improve outcomes. We have previously shown that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in human FLT3-ITD AML leukemia stem cells (LSC), which demonstrate increased sensitivity to the small molecule SIRT1 inhibitor Tenovin-6 (Li at al., Cell Stem Cell 15, 431, 2014). We have now determined that Tenovin-6, in addition to SIRT-1 inhibition and activation of p53 activity, also functions as an effective inhibitor of autophagic flux, mediated by an aliphatic tertiary amine side chain that mediates alkalization of lysosomes. Comparison of Tenovin (TV) analogs with or without SIRT1 or autophagy inhibitory properties revealed that TVs can kill melanoma cells independently of SIRT1 inhibition and that autophagy blockade correlates with ability to completely eliminate cancer cells. Autophagy is known to promote survival and facilitate tumorigenesis and drug resistance by degradation and recycling of intracellular proteins and organelles in lysosomes. In the present work, we investigated the roles of SIRT1 inhibition and autophagy inhibition in targeting AML cells. The TV analogs studied includedTV-39 (inhibits both SIRT1 and autophagy), TV-39OH (inhibits SIRT1 alone), TV-50 (inhibits autophagy alone), and TV-50OH (inhibits neither). We observed that FLT3-ITD + AML cell lines (MV4-11 and MOLM13) demonstrated increased sensitivity to TV analogs with either autophagy inhibitory (TV-50) or SIRT1 inhibitory (TV39OH) effects compared with FLT3-WT cell lines (OCI-AML3 and KG1A). The combination of TV50 and TV39OH resulted in synergistic cytotoxicity in FLT3-ITD+ AML cell lines, as well as in primary AML CD34+cells bearing the FLT3-ITD mutation (Table 1). Western blot analysis indicated that FLT3-ITD+ cells treated with TV39 showed accumulation of LC3B-II, consistent with blockade of a late stage of autophagy, as well as increased acetylated and total p53 expression, consistent with SIRT1 inhibition. Cells treated with TV39OH showed increased acetylated and total p53 without change of LC3B-II levels, whereas cells treated with TV50 alone showed only increase of LC3B-II and not in p53 levels. Knockdown (KD) of p53 using lentivirus-expressed shRNA constructs reversed the inhibitory effects of TVs with both SIRT1 and autophagy inhibitory properties. The combination of the FLT3 TKI AC220 with either TV39OH or TV50 resulted in synergistic cytotoxicity in MV4;11 and Molm-13 cells, indicating that these agents could induce enhance elimination of FLT3-ITD+ cells by TKIs. To confirm the interaction of SIRT1 and autophagy inhibition in targeting FLT3-ITD AML cells, we knocked down ATG5 and SIRT1 expression in MV4-11 and Molm13 cells using lentivirus expressed shRNA. KD of either ATG5 or SIRT1 individually resulted in significant induction of apoptosis and inhibition of proliferation in AML FLT3-ITD cell lines compared with shRNA controls (Table 2). The combined knockdown of SIRT1 and ATG5 induced further increase in apoptosis compared to KD of individual genes. KD of SIRT1 or ATG5 significantly enhanced levels of ROS in FLT3-ITD positive cell lines, measured using the CellROX® Deep Red Reagent (shCtrl vs. shATG5+shSirt1, 1 vs1.74 p=0.02; shCtrl vs shATG5, 1vs 1.83, p=0.009; shCtrl vs. shATG5, 1 vs. 1.85 p=0.008). In an ongoing experiment, we are checking the effect of KD of SIRT1, ATG5 or of both SIRT1 and ATG5 in Molm13 cells in their growth in NSG mice. We conclude that combined inhibition of SIRT1 activity and autophagic flow synergistically targets FLT3-ITD+ AML cell lines and primary CD34+ cells, and enhances their targeting by FLT3 kinase inhibitors. Combined targeting of autophagic flow and SIRT1 activity appears to be a promising strategy to enhance elimination of FLT3-ITD+ AML stem cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunoproteasome Inhibition to Target AML with Activated RAS Pathways

Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the only leukemia cells with self-renewal potential and ability to recapitulate the disease. Our goal is to develop therapeutics that target and eradicate AML LSCs. We showed that activated NRAS facilitates self-renewal in a murine model of AML; mTORC1 activation by NRAS is important for this effect (Sachs et al. Blood 2014). RAS activates mTORC1 through the PI3K/AKT/mTOR pathway which is highly upregulated in AML (Xu et al. Blood 2003, Altman et al. Oncogtarget 2011). Recently, we found that RAS-activated malignancies, including AML, induce immunoproteasome formation via mTORC1 (Yun et al. Mol Cell 2016). Pan-proteasome inhibitors, such as bortezomib and carfilzomib, are widely used in the treatment of lymphoid malignancies and have some activity in AML. We hypothesize that specific inhibition of the immunoproteasome may target NRAS-mediated self-renewal in LSCs. We use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). We investigated the possible role of the immunoproteasome in our murine model and found that abolishing NRASG12V transgene expression led to a loss of immunoproteasome component genes expression (p<0.001). We also found that the LSC-enriched subpopulation preferentially upregulated expression of immunoproteasome component genes (p<0.002) in comparison to non-stem cells from this model. Accordingly, we found that primary human AML CD34+ precursors upregulated immunoproteasomal components relative to CD34- AML cells by up to 3.7 fold. mTOR inhibition with everolimus diminished expression of immunoproteasome component genes (p=0.05) suggesting that immunoproteasome activity may be NRASG12V/mTOR-dependent in this model as well. Next, we sought to identify the self-renewing cells among immunophenotypically defined LSC-enriched subgroup in this model. We performed single-cell RNA sequencing on the LSC-enriched population from this model. We identified three discrete trasncriptional profiles among the LSCs. Comparing the single-cell transcriptional profiles of NRASG12V-expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of these profiles are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted LSCs based on CD36 and CD69 expression as well as established immunophenotypic markers, Mac1, Kit, Sca-1 (LSC-enriched supopulation in this model is Mac1LowKit+Sca1+). These sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that CD36-CD69+Mac1LowKit+Sca1+ cells (consistent with Profile 1 gene expression) rapidly transferred leukemia with high penetrance. In contrast, CD36+CD69- Mac1LowKit+Sca1+ cells (consistent with Profile 2 gene expresison) did not transfer leukemia to mice (p < 0.004). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature at the singel-cell level and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. We performed single-cell RNA sequencing on CD34+ human AML precursors obtained from a diagnostic bone marrow specimen. Analogous to our murine model, we found that these human AML cells express 2 distinct single-cell transcriptional profiles and that differentially express RAS-activated gene expression profiles. Finally, we found that, PR957, a specific immunoproteasome inhibitor, inhibits in vitro colony formation more effectively than carfilzomib (a second generation pan-proteasome inhibitor) in our murine model of AML suggesting that immunoproteasome inhibition may be specifically target self-renewal in AML. In these experiments, we use single-cell RNA sequencing to identify the self-renewing subset of RAS-driven LSCs at the single-cell level and implicate immunoproteasome inhibition as an approach to target RAS-mediated self-renewal. DisclosuresNo relevant conflicts of interest to declare.

Sunitinib Reduces Acute Myeloid Leukemia Clonogenic Cells &lt;i&gt;in Vitro&lt;/i&gt; and Has Potent Inhibitory Effect on Sorted AML ALDH+ Cells

Sunitinib is an orally administered, multi-target tyrosine kinase inhibitor that has been approved by the FDA for the treatment of renal cell carcinoma and imatinib resistant gastro-intestinal tumors. Anti-leukemic activity of sunitinib has been examined in early clinical trials with limited success. However, recent trials on acute myeloid leukemia (AML) patients carrying FLT3 mutations have shown promising results. Effects of sunitinib on leukemic clonogenic cells and potential leukemic stem cells have not been examined so far. We analyzed the anti-proliferative and apoptotic properties of sunitinib on AML-derived cell lines. We also tested the effect of sunitinib on AML patient derived clonogenic cells (AML-CFC), as well as flow-sorted potential leukemic progenitors. Peripheral blood or bone marrow samples were obtained from newly diagnosed AML patients and flow sorted for CD34+ CD133+ or ALDH+ cells. Umbilical cord blood derived CD34+ cells were used as normal controls. Sunitinib induced growth arrest and apoptosis in AML derived cell lines. In addition, 7 μM sunitinib induced 75% reduction of AML-CFC as compared to DMSO treated control (±6.79%; n = 4). In contrast, 7 μM sunitinib treatment of umbilical cord blood derived normal CD34+ cells showed 29% reduction in AML-CFC (±6.77%; n = 5). Treatment of ALDH+ cells sorted from 2 AML cases and CD34+ CD133+ cells from one patient showed reduction of AML-CFC on treatment with sunitinib. Our study highlighted a potent anti-proliferative and proapoptotic effect of sunitinib on AML cell lines, AML patient derived clonogenic cells and potential leukemic stem cells.

The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation

The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21CIP1. Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34+ progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34+ progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34+ cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21CIP1 mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21CIP1 expression during APL differentiation.

Targeting Autophagy As a Potential Therapeutic Strategy for the Treatment of AML

Autophagy is a catabolic process whereby redundant organelles and proteins are digested and recycled to maintain cellular homeostasis. Malignant cells might activate autophagy in response to cellular stress, and/or to provide metabolites required for rapid cell proliferation. In addition, elevated autophagy is associated with increased chemoresistance. In normal hematopoiesis, autophagy is important for stem and progenitor cell (HSPC) maintenance. We previously observed that basal autophagy is higher in HSPCs compared more differentiated cells. Genetic interference with the autophagy machinery resulted in a strong reduction in HSPC frequencies, without interfering with the differentiation program.To study the role of autophagy in acute myeloid leukemia (AML), steady-state autophagy was measured in a panel of different myeloid leukemic cell lines (n=8). The autophagic flux was determined by the relative accumulation of cyto-ID (a marker for autophagic vesicles) measured by flow cytometry after hydroxychloroquine (HCQ) or Bafilomycin-1A treatment and LC3-II/p62 accumulation by Western blotting. Interestingly, in leukemic cell lines, a higher basal autophagy flux correlated with increased sensitivity to prolonged inhibition (6 days) of autophagy with different concentrations of HCQ (5 uM R2 =0.52 and 20 uM R2 =0.76), while a mild reduction in expansion was observed in cord blood (CB) derived normal CD34+ cells, after treatment with HCQ (5 uM). In line with the leukemic cell lines, primary sorted AML CD34+ cells (n=36) also showed decreased survival upon HCQ (20 uM) treatment compared to normal bone marrow CD34+ cells (N=6; NBM CD34+: 41.7% ± 7.1 vs. AML CD34+: 21.3% ± 3.2, p=<0.05).Gene expression analysis of autophagy genes by qPCR revealed increased expression of essential autophagy genes ATG5 and ATG7 in primary AML CD34+ compared to NBM CD34+ cells (AML vs. NBM: ATG5 10.3 fold increase, p=<0.05; ATG7 5.9 fold increase, p=<0.01). Therefore, to study the functional consequences in AML, ATG5 and ATG7 were downregulated in leukemic cell lines and primary AML CD34+ cells (n=6), using lentiviral shRNAs. Significant downregulation of ATG5 and ATG7 was confirmed by qPCR, which was also sufficient to inhibit the autophagy flux as demonstrated by reduced accumulation of LC3-GFP puncta. In line with results of HCQ treatment, downmodulation of ATG5 and ATG7 resulted in a strong reduction in expansion of leukemic cell lines. Also, transduced primary AML blasts cultured on MS5 bone marrow stromal cells for 4 weeks showed a distinct reduction in expansion (shSCR vs. shATG5 2.1 fold reduction, p=<0.05; shATG7 2.5 fold reduction p=<0.01).The genetic or pharmaceutical inhibition of autophagy triggered an apoptotic response in leukemic cellsas determined by Annexin V measurements. This coincided with increased expression of p53 and the downstream pro-apoptotic target genes BAX and PUMA and the putative AKT inhibitor PHLDA3. Moreover, HCQ induced apoptosis could be rescued by downregulation of p53 by RNAi in leukemic cell lines that express wild-type P53. Prolonged exposure to HCQ did also result in an apoptotic response in leukemic cell lines harboring p53 mutations, but without the upregulation of BAX, PUMA and PHLDA3.In conclusion, our results indicate that AML CD34+ cells are highly dependent on the autophagy machinery for growth and cell survival. Targeting autophagy might provide a new therapeutic option for treatment of this type of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

A New Strategy to Target Acute Myeloid Leukemia Stem and Progenitor Cells Using Chidamide, a Histone Deacetylase Inhibitor

Leukemia stem cells (LSCs) are responsible for treatment failure and relapse in acute myeloid leukemia (AML). Therefore, development of novel LSCs-targeting therapeutic strategies is of crucial clinical importance to improve the treatment outcomes of AML. Histone deacetylase (HDAC) inhibitors have emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. HDAC inhibitors have shown potent and specific anticancer stem cell activities in preclinical studies. Chidamide, a novel benzamide-type selectively HDAC inhibitor, has been reported to induce G1 arrest and apoptosis in the relatively mature progenitor population, whereas its effect on primitive LSCs has not been clarified. In this study, we demonstrated that chidamide specifically induces apoptosis in LSC-like cells and primary AML CD34+ cells in a concentration- and time-dependent manner. Our further molecular mechanistic study uncovered that chidamide induces LSCs death by activation of reactive oxygen species (ROS). It compromises the mitochondria membrane potential, modulates anti-apoptotic and pro-apoptotic proteins in BCL2 family and activates caspase-3 leading to PARP degradation. Meanwhile, chidamide activates CD40 and modulates its downstream signaling pathways, JNK and NFκB. The results of this study suggest that chidamide may be a novel LSC-targeting agent for AML therapeutics. DisclosuresNo relevant conflicts of interest to declare.

P38MAPK Inhibition Blocks Inflammatory Signaling in Acute Myeloid Leukemia

Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients.Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 < 1000nM were considered drug responsive.Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity.Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. DisclosuresWinski:Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

BH3 mimetic ABT-737 induces apoptosis in CD34+ acute myeloid leukemia cells and shows synergistic effect with conventional chemotherapeutic drugs.

Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease. The early immature CD34+ AML cell subpopulation is frequently impervious to intensive chemotherapy, making them largely responsible for relapse of AML. CD34+ AML cells have higher level of Bcl-2 protein expression than the CD34- subpopulation. As such, development of drugs that specifically target the Bcl-2 may have the potential to eliminate immature CD34+ AML progenitor cells and provide therapeutic benefit. In this work, we made an attempt to investigate the cytotoxic effect of a novel Bcl-2 family inhibitor, ABT-737, on CD34+ AML cell lines (KG1a and Kasumi-1) as well as CD34+ primary AML cells. Primary human CD34+ cells were isolated from bone marrow mononuclear cells using CD34 MicroBead kit. The growth inhibitory effect was measured by cell counting kit-8. Apoptosis was analyzed by annexin V/PI assays. Protein expression was determined by Western blotting analysis. Inhibition of Bcl-2 by ABT-737 effectively inhibited growth and induced apoptosis in CD34+ AML cell lines and CD34+ primary AML cells without affecting CD34+ normal hematopoietic cells. Furthermore, Western blot analysis showed that ABT-737 induced apoptosis associated with caspase-3 activation and poly ADP-ribose polymerase (PARP) degradation. Finally, ABT-737 synergistically enhanced the cytotoxic effect of cytarabine and daunorubicin in CD34+ AML cells. Taken together, these findings indicate that ABT-737 may offer as a promising molecular targeting agent in CD34+ AML.

HDAC8 Inhibition Specifically Targets Inv(16) Acute Myeloid Leukemic Stem Cells by Restoring p53 Acetylation

Acute myeloid leukemia (AML) is driven and sustained by leukemia stem cells (LSCs) with unlimited self-renewal capacity and resistance to chemotherapy. Mutation in the TP53 tumor suppressor is relatively rare in de novo AML; however, p53 can be regulated through post-translational mechanisms. Here, we show that p53 activity is inhibited in inv(16)(+) AML LSCs via interactions with the CBFβ-SMMHC (CM) fusion protein and histone deacetylase 8 (HDAC8). HDAC8 aberrantly deacetylates p53 and promotes LSC transformation and maintenance. HDAC8 deficiency or inhibition using HDAC8-selective inhibitors (HDAC8i) effectively restores p53 acetylation and activity. Importantly, HDAC8 inhibition induces apoptosis in inv(16)(+) AML CD34(+) cells, while sparing the normal hematopoietic stem cells. Furthermore, in vivo HDAC8i administration profoundly diminishes AML propagation and abrogates leukemia-initiating capacity of both murine and patient-derived LSCs. This study elucidates an HDAC8-mediated p53-inactivating mechanism promoting LSC activity and highlights HDAC8 inhibition as a promising approach to selectively target inv(16)(+) LSCs.

Developing therapeutic strategies targeting poor prognosis acute myeloid leukemia

Development of various anti-cancer agents has allowed the majority of acute myeloid leukemia (AML) patients to achieve complete remission. Nevertheless, leukemia patients with poor prognostic factors succumb to the disease. We have aimed to understand the in vivo biology of human AML cells and to create therapeutic strategies targeting poor prognosis AML. To this end, we developed an in vivo model for human AML by injecting primary AML cells into NSG newborns via the superficial temporal vein. Immunohistochemical staining for human cells revealed that injected CD34+CD38- AML cells preferentially home to endosteal region of the recipient bone marrow. CD34+CD38- AML cells generated CD34+CD38+ cells and CD34- cells in the bone marrow and spleen over time, recapitulating phenotypic heterogeneity of AML. Flow cytometric analyses and immunofluorescent studies demonstrated that CD34+CD38- cells stay cell cycle quiescent near endosteum, leading to resistance to cell cycle-dependent cytotoxic agents. To identify therapeutic targets in chemotherapy-resistant AML cells, we performed global gene expression profiling using AML CD34+CD38- cells and normal CD34+CD38- cells. Through the comparison, we found that HCK is over-represented in AML compared with normal HSCs. We then screened compounds that can inhibit HCK at lower concentrations in a mobility shift assay. Based on the structural analysis of hit compounds, we synthesized a molecule, RK-20449, that can form multiple hydrogen bonds with ATP binding site of HCK. In vitro experiments demonstrated that the compound was effective particularly against FLT3-ITD+ AML cells. Finally, we confirmed that RK-20449 targeted human AML cells in NSG mice engrafted with patient-derived AML cells with FLT3-ITD mutation. Based on the findings, we further aim to develop RK-20449 as a therapeutic agent in the future. Development of various anti-cancer agents has allowed the majority of acute myeloid leukemia (AML) patients to achieve complete remission. Nevertheless, leukemia patients with poor prognostic factors succumb to the disease. We have aimed to understand the in vivo biology of human AML cells and to create therapeutic strategies targeting poor prognosis AML. To this end, we developed an in vivo model for human AML by injecting primary AML cells into NSG newborns via the superficial temporal vein. Immunohistochemical staining for human cells revealed that injected CD34+CD38- AML cells preferentially home to endosteal region of the recipient bone marrow. CD34+CD38- AML cells generated CD34+CD38+ cells and CD34- cells in the bone marrow and spleen over time, recapitulating phenotypic heterogeneity of AML. Flow cytometric analyses and immunofluorescent studies demonstrated that CD34+CD38- cells stay cell cycle quiescent near endosteum, leading to resistance to cell cycle-dependent cytotoxic agents. To identify therapeutic targets in chemotherapy-resistant AML cells, we performed global gene expression profiling using AML CD34+CD38- cells and normal CD34+CD38- cells. Through the comparison, we found that HCK is over-represented in AML compared with normal HSCs. We then screened compounds that can inhibit HCK at lower concentrations in a mobility shift assay. Based on the structural analysis of hit compounds, we synthesized a molecule, RK-20449, that can form multiple hydrogen bonds with ATP binding site of HCK. In vitro experiments demonstrated that the compound was effective particularly against FLT3-ITD+ AML cells. Finally, we confirmed that RK-20449 targeted human AML cells in NSG mice engrafted with patient-derived AML cells with FLT3-ITD mutation. Based on the findings, we further aim to develop RK-20449 as a therapeutic agent in the future.

A New Strategy to Target Acute Myeloid Leukemia Stem and Progenitor Cells Using Chidamide, a Histone Deacetylase Inhibitor.

Leukemia stem cells (LSCs) are responsible for treatment failure and relapse in acute myeloid leukemia (AML). Therefore, development of novel LSCs-targeting therapeutic strategies is of crucial clinical importance to improve the treatment outcomes of AML. Histone deacetylase (HDAC) inhibitors have shown potent and specific anticancer stem cell activities in preclinical studies. Chidamide, a novel benzamide-type selectively HDAC inhibitor, has been reported to induce G1 arrest and apoptosis in the relatively mature progenitor population, whereas its effect on primitive LSCs has not been clarified. In this study, we demonstrated that chidamide specifically induces apoptosis in LSC-like cells and primary AML CD34(+) cells in a concentration- and time-dependent manner. Our further molecular mechanistic study uncovered that chidamide induces LSCs death by activation of reactive oxygen species (ROS). It compromises the mitochondria membrane potential, modulates antiapoptotic and pro-apoptotic proteins in BCL2 family and activates caspase-3 leading to PARP degradation. Meanwhile, chidamide activates CD40 and modulates its downstream signaling pathways, JNK and NFκB. The results of this study suggest that chidamide may be a novel LSC-targeting agent for AML therapeutics.

Abstract 1691: SL-401 activity against AML blasts correlates with CD123 levels and does not down-regulate CD123 expression

Abstract Despite the progress, the current therapies for acute myeloid leukemia (AML) are not curative partly due to failure to eradicate the leukemic stem cells (LSCs) that repopulate the leukemia. CD123 is the alpha-chain of interleukin-3 receptor (IL-3R) that is expressed on AML and AML LSCs, while it is undetectable in normal hematopoietic stem cells. We report here time and dose dependent cytotoxic activity of SL-401(Stemline Therapeutics, Inc), a diphtheria toxin interleukin-3 fusion protein which is designed to selectively bind, internalize and induce apoptosis of CD123+ primary AML cells isolated from patients. AML blasts were obtained from leukapheresis of patients and cultured in RPMI1640 with 20% fetal bovine serum and antibiotics and supplemented with growth factors GM-CSF and SCF (10ng/ml) overnight before treatment with increasing concentrations of SL-401. CD123 expression of AML blasts was analyzed using multicolor flow cytometry before and 24 hours after treatment with SL-401. The viability of the cells before and after treatment with increasing concentrations of SL-401 was measured by annexinV-FITC and propidium iodide staining. SL-401 induced dose-dependent (SL-401: 10, 100, 1000ng/ml) cytotoxicity in primary AML in nanomolar range (trend p = 0.0002 at 48hr), which correlated with surface expression levels of CD123. AML CD34+CD33+ blasts showed high CD123 expression levels and were susceptible to cytotoxicity with SL-401 (P&amp;lt;0.0001,1000ng/ml at 48hr). CD34−CD33+ committed leukemic cells were also CD123+ and responded to SL-401. To determine if SL-401 modulated CD123 levels after exposure, AML blasts pre-treated with SL-401 for 24hr were analyzed by flow cytometry for receptor expression. Importantly, SL-401 treatment after 24 hours did not down-regulate CD123 expression on primary AML blasts indicating a lack of an escape mechanism for malignant cells. These results provide evidence that time- and dose-dependent activity of SL-401 against CD123+ AML blasts correlates with CD123 expression. Moreover, treatment does not down-regulate IL-3R expression. Clinical studies of SL-401 in patients with relapsed/refractory AML with evaluation of CD123 expression and blastic plasmacytoid dendritic cell neoplasm (BPDCN) are ongoing. Citation Format: Rajeswaran Mani, Bhavani Gopalakrishnan, Xiaokui Mo, Chris Brooks, William Blum, Gerard Lozanski, Sumithira Vasu, Natarajan Muthusamy. SL-401 activity against AML blasts correlates with CD123 levels and does not down-regulate CD123 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2015-1691

Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs.In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. DisclosuresNo relevant conflicts of interest to declare.