Primary Acute Myeloid Leukemia CD34 Research Articles

Synergistic Activity of Adefovir Dipivoxil and Venetoclax to Target the Bioenergetic Metabolism of Acute Myeloid Leukemia

Adefovir dipivoxil (ADV) is an oral prodrug of the nucleotide analogue adefovir that inhibits viral DNA polymerase and is an FDA-approved drug for the treatment of hepatitis B virus. Preliminary evidence of the antileukemic activity of ADV was recently reported in a patient with acute promyelocytic leukemia (APL; Gong, X., et al. Exp Hematol Oncol, 2022). Herein we report on the antileukemic activity of ADV, alone and in combination with venetoclax (VEN), an oral FDA-approved BCL-2 inhibitor for the treatment of acute myeloid leukemia (AML). Firstly, a significant dose-dependent reduction of cell growth and increase in apoptosis were observed in FLT3-ITD+ MV4-11 (IC50: 97nΜ) and Molm-13 (IC50: 0.816μM) cells, and in primary CD34+ blasts from refractory/relapsed (R/R) AML patients (n=5; IC50: 1.285-3.703μM), exposed to serial concentrations of ADV for 48 hours (hrs). In contrast, no significant changes were observed in ADV-treated CD34+ cells from healthy donors (n=5). Combination of ADV and VEN were synergistic (combination index < 1.0) in reducing cell viability of AML cells (i.e., MV-4-11, U937, KG1 and THP-1) and CD34+ blasts from R/R AML patients (n=3). To gain insights into the molecular mechanisms underlying the antileukemic activity of ADV alone and in combination with VEN, we performed RNA-Seq on AML CD34+ blasts from R/R AML patients (n=6) treated with vehicle, ADV (5 μΜ), VEN (100 nΜ), or ADV+VEN for 24 hrs. Gene set enrichment analysis (GSEA) highlighted upregulation of p53 pathway and apoptosis signaling, and downregulation of oxidative phosphorylation (OXPHOS), glycolysis, and MYC target hallmark gene sets in ADV- and ADV+VEN-treated cells compared with vehicle-treated controls. These results were confirmed by western blot analysis showing a dose-dependent reduction in c-MYC, anti-apoptotic proteins (i.e., BCL-2 and MCL-1), and carnitine palmitoyltransferase-1a [CPT1A, a key enzyme for fatty acid oxidation (FAO), a process that feeds into OXPHOS], and an increase in pro-apoptotic proteins (i.e., BAX and BAK) in ADV- and ADV+VEN-treated MV4-11 and primary AML CD34+ cells, compared with vehicle-treated control cells. Seahorse analysis confirmed a significant reduction of OXPHOS [measured by oxygen consumption rate (OCR)], associated with decreased mitochondrial numbers and sizes evaluated by electron microscopy imaging of AML CD34+ blasts treated with ADV or ADV+VEN compared with vehicle-treated controls. To test in vivo the antileukemic activity of ADV+VEN, we treated a cohort of CD45.1 recipient mice transplanted with BM cells from congenic CD45.2 Mll PTD/WT/Flt3 ITD/ITD AML mice with vehicle, ADV (100mg/kg, oral gavage), VEN (100mg/kg, oral gavage), or ADV+VEN (n=10 per group) for 4 weeks. ADV+VEN-treated mice lived longer (median survival: 60 days after treatment completion) than ADV (20 days, p=0.0001), VEN (20 days, p=0.0001), or vehicle (14 days, p=0.0002) -treated controls (Figure 1). We then performed secondary transplants by engrafting 10 6 BM cells from treated donors into CD45.1 recipient mice (n=10 per group). Recipients of BM from ADV+VEN-treated donors survived longer (median survival: 83 days) than recipients of BM from ADV (60.5 days, p<0.01), VEN (45.5 days, p<0.0001), and vehicle (42.5 days, p<0.0001) -treated donors (Figure 2), confirming a decrease in leukemic stem cell (LSC) burden by ADV+VEN. Next, we examined the antileukemic activity of ADV+VEN in human AML models. We first treated a cohort of NSG mice (n=10 per group) transplanted with luciferase-expressing Molm-13 cells. ADV+VEN-treated mice survived longer (median survival: 59.5 days) than ADV (52 days, p=0.04), VEN (40 days, p<0.0001), and vehicle (40.5 days, p<0.0001) -treated mice. We then treated a cohort of NSG mice (n=8 per group) transplanted with FLT3-ITD+ blasts from a patient with R/R AML. ADV+VEN-treated mice survived longer (median: 38.5 days) than ADV (34.5 days, p=0.035), VEN (25 days, p<0.0001), and vehicle (18.5 days, p<0.0001) -treated mice. In summary, ADV has in vivo anti-leukemic activity in AML likely by interfering with the bioenergetic metabolism of leukemic cells and decreasing their apoptotic threshold. The combination of ADV and VEN is seemingly synergistic and prolonged survival of murine and human AML models, providing a rationale of translating this “all oral” regimen to the clinic for treatment of AML patients, especially those requiring low intensity therapy.

KDM6A Modulates Anti-Tumor Immune Response By Integrating Immunogenic Cell Death in Human Acute Myeloid Leukemia

Identifying tumor cell-epigenetic regulation of immune activation in acute myeloid leukemia (AML) would be instrumental in developing next-generation of targeted therapy. AML cells have low mutational burden; inefficient ‘cross-presentation’ through conventional dendritic cells (DC) and immune activation are the major challenges for AML immunomodulation. ‘Immunogenic cell death’ (ICD) represents changes in the composition of the tumor cell surface as well as the release of ‘damage associated molecular patterns’ (DAMPs) in the tumor microenvironment that stimulates the dysfunctional immune system. DAMPs in turn operate on a series of receptors expressed by DCs and natural killer (NK) cells and contribute to long-lasting, protective anti-tumor immunity. For example, plasma membrane exposure of endoplasmic-reticulum (ER)-translocated calreticulin (ecto-CRT), which functions as an ‘eat me’ signal, is an important hallmark of ICD. Previous study suggested that calreticulin (CALR) promotes immunity and type I interferon-dependent survival in mice with AML (Fucikova, Blood 2016; Chen, Oncoimmunology 2017). In addition, ecto-CRT on malignant blasts correlated with improved NK cell-mediated cytotoxicity in AML patients with superior overall survival (Truxova, Haematologica 2020). Ecto-CRT has also been identified as an endogenous sterile ligand for NK cells (Sen Santara, Nature 2023). Nevertheless, molecular regulation of ICD in the context of epigenetic derangements and inflammation and its implication in AML immunotherapeutic modulation is poorly understood. KDM6A is a histone 3 lysine 27 demethylase that plays tumor suppressor function in AML (Gozdecka, Nat Genet 2018; Sera, Blood 2021). KDM6A escapes X-chromosome inactivation, and loss-of-function deletion or point mutations associate with resistance to ‘3+7‘ chemotherapy (Stief, Leukemia 2020). We have identified that in comparison to control AML cells, deficiency of KDM6A ( KDM6A-kd) associated with daunorubicin/etoposide-induced ICD, which was manifested by significant increase (2-3 fold , P<0.05) in ecto-CRT, plasma membrane exposure of pE1F2α, HSP70 and HSP90 with extracellular release of HMGB1 and ATP. Similarly, pharmacological inhibition of JmjC catalytic function using GSK-J4 sensitized human AML lines as well as primary AML patient bone marrow-derived CD34 + hematopoietic stem/progenitor cells to ICD. Induction in ICD was accompanied by an elevated type I interferon production that was functionally active in promoting interferon regulated gene expression. Interestingly, treatment with acute recombinant IFN-β caused an increase in activated STAT1 and inflammatory gene expression in KDM6A-kd AML cells compared to control AML. Corroborating these results, gene set enrichment analysis revealed a significant enrichment (NES=1.708, P=0.007) of upregulated type I interferon signatures (n=40) in Kdm6a -/- primary leukemia cells. Re-analysis of ChIP-seq results indicated 1647 Kdm6a occupied genes, which were upregulated upon Kdm6a loss. Gene ontology (GO) analysis of the 1647 genes further suggested an enrichment ( P<0.001) of type I interferon-responsive GO terms, indicating Kdm6a role in interferon signaling. Ecto-CRT level relies on premortem ER stress. In OHSU AML, KDM6A expression correlates with ER stress/unfolded protein response. Mechanistically, quantitative-ChIP and promoter-reporter assays indicated that KDM6A-kd de-repressed ICD induced expression of ATF4 and HSPA5 through chromatin and transcriptional remodeling. In agreement with these findings, AML blasts-healthy immune cells ex vivo co-culture experiments revealed that increase in ICD in KDM6A-kd AML was associated with activation (~2-foldincrease inCD80 +CD86 +, P<0.05) and phagocytic function of DCs. Loss of KDM6A was shown to bestow ‘BRCAness’ (Boila, Leukemia 2023), and PARP inhibition itself can cause ICD and augment tumor immunity. We observed that there was a significant induction in ICD and expression of NKG2D ligands ULBP1, ULBP5 in KDM6A-kd AML cells treated with olaparib. In addition, olaparib and GSK-J4 treatment showed additive effects in ICD and immune activation using primary AML CD34 + cells. Together, we illustrate that KDM6A regulates ICD through epigenetic mechanisms, while KDM6A deficient AML subtypes could be sensitized with immunomodulatory targeted therapy.

Addiction to Small Gtpase ARF6-Mediated Sphingolipid Homeostasis By AML but Not the Host

Background Cancer cells' unique metabolic demand has emerged as a promising therapeutic target in Acute Myeloid Leukemia (AML). The vesicular trafficking and signaling gatekeeper, ADP-Ribosylation Factor 6 (ARF6), belongs to the RAS superfamily of small GTPases and has been shown to regulate the homeostasis of lipids such as PIP2, phosphatidic acids, and cholesterol. Here we describe AML cells' unusual addiction to ARF6-mediated sphingolipid homeostasis and its clinical significance. Experiments and Results ARF6 knockdown (KD) or CRISPR-mediated ARF6 knockout (KO) inhibited the proliferation of AML cell lines, THP-1 and MV4-11, as well as the colony formation of primary AML CD34 + cells. Surprisingly, Tamoxifen-induced whole body KO of Arf6 in adult Arf6 f/f;Rosa-CreERT 2 mice led to healthy and grossly normal mice with a normal lifespan. Adult mice lacking ARF6 show no abnormality in complete blood count, urine test, or histology. Hence, ARF6 is genetically indispensable for AML, but not for the host. To confirm AML's addition to ARF6 in vivo, we introduced Arf6 KO in an MLL-AF9-induced AML model in C57BL6/J mice. We are currently following up with the disease progression between Arf6-KO AML group and control AML group, and expect to obtain results before the 2023 ASH meeting. After discovering that ARF6 is genetically necessary for AML but not needed for the host, we analyzed its clinical significance. Since ARF6 mutations are not detected in AML samples, we focused on analyzing its expression. With univariate analysis, we show that high ARF6 expression (top 50%) is significantly associated with worse overall survival (OS) and worse disease-free survival (DFS) of AML patients in the TCGA-NEJM-2013 (TCGA) cohort (N=200) and the Münchner Leukämielabor (MLL) cohort (N=579). In multi-variate analysis, high ARF6 expression is significantly associated with worse OS in the BeatAML cohort (N=562) and shows a trend towards worse OS and DFS in the TCGA cohort. Following a hypothesis-driven approach, we investigated the role of ARF6 in regulating sphingolipid homeostasis by HPLC-MS lipidomic analyses of AML cells (THP-1) with or without ARF6 depletion. Lipidomic profiles revealed that ARF6 KD led to an increase of the overall ceramide/sphingomyelin ratio, suggesting a defect in sphingolipid homeostasis. We then analyzed the expression of sphingolipid metabolic enzymes and observed that ARF6 KD downregulated sphingomyelin synthase 1 and 2 (SGMS1 and SGMS2) expression, hindering ceramide-to-sphingomyelin conversion. Supporting this, we found a positive linear correlation between ARF6 and SGMS1/2 expression among the patients in TCGA cohort, confirming their correlation. High expression of the ARF6 effectors SGMS1/2 is also associated with worse OS and DFS in the TCGA cohort and the MLL cohort in univariate analysis, resembling that of ARF6. Downstream of ceramide accumulation, ARF6 KD inhibited the pro-survival AKT pathway, confirming the pro-apoptotic roles of ceramide in cancers. The anti-proliferative effect of ARF6 depletion can be rescued by sphingomyelinase inhibitor GW4869 (blocking ceramide production) or by expressing a constitutively active form of AKT (AKT1 E17K). To pharmacologically target ARF6, we developed two generations of ARF6 small molecular inhibitor pro-drugs, named NAV-2729 and A6-4471. NAV-2729 and A6-4471 showed high specificity to ARF6 without inhibiting other small GTPases, with in vitro IC 50 in the lower μM range. Both NAV-2729 and A6-4471 inhibited AML cell line proliferation and reduced primary AML cell colony formation in lower μM range. Cord blood normal CD34 + cells are not sensitive to ARF6 inhibitors, but non-specific toxicity was observed at higher concentrations. When tested in vivo with THP-1 xenografts in NSG mice, both inhibitors reduced circulating AML cells without obvious toxicity to the host. Conclusion ARF6 is necessary for AML proliferation and its higher expression is associated with worse patient survival. However, ARF6 is dispensable for normal host physiology. Mechanistically, ARF6 maintains critical ceramide/sphingomyelin balance in AML by regulating SGMS1/2 expression. Pharmacologically inhibiting ARF6 suppressed AML proliferation in vitro and in vivo in a genotype-agnostic fashion. Our study implicates ARF6 as a potentially actionable and safe target in AML.

PNC¯27 Targeting Plasma Membrane HDM2: A Potentially Novel Therapeutic Approach for Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy. The vast majority of AML patients are immediately refractory to induction therapy or subsequently relapses following transient complete remission, due to persistence of treatment-resistant leukemia stem cells (LSC). Thus, there is a clear need to identify new targets and develop novel and effective therapeutic approaches in this population. HDM2, an E3 ubiquitin-protein ligase, binds to p53 tumor suppressor in the cytoplasm, repressing p53 transcriptional activity and facilitating its proteasomal degradation. Overexpression of HDM2 plays a key role in tumorigenesis. Recently, it has been reported that HDM2 is not only localized to the cytoplasm but can also be found highly expressed on the plasma membrane of various cancer cells (i.e., colon, breast, pancreas) but not on normal counterparts (PNAS 2010 :107(5):1918-23). PNC-27(Oncolyze, Inc.), a 32-amino acid synthetic peptide with a penetratin domain and a HDM-2-binding domain of p53, can bind with cell membrane HDM2, causing membrane pore formation, thus inducing necrosis in solid tumor cells. However, the activity of PNC-27 in AML has not been established. Using flow cytometry analysis and confocal microscopy with anti-HDM2 antibody (Santa Cruz,N-20), we showed that HDM2 was expressed on the membrane of LSC-enriched CD34+CD38- primary blast populations procured from consecutive AML patients (n=6) at levels greater than those observed on normal CD34+CD38- cells [hematopoietic stem cells (HSC)] from healthy individuals (n=12) [median fluorescence intensity (MFI) (HDM-2/IgG control) 2.5±0.6 vs 1.1±0.1; p<0.01)]. Similar results were also observed in AML cell lines (i.e., THP-1, MV4-11,MOLM-13, NOMO1).Further, we showed that exposure to PNC-27 (50-200ug/ml) for 72 hours resulted in significantly reduced viability and colony formation activity (P<0.001) in both AML cell lines and human AML CD34+ cells compared with normal CD34+ cells, in a dose- and time-dependent manner. PNC-27 antileukemia activity was significantly and directly correlated with cell membrane HDM2 expression levels (p<0.05), more HDM2 expression, higher PNC-27 efficacy. Confocal microscopy using fluorescent anti-HDM2 and anti-PNC-27 antibodies showed that PNC-27 and HDM-2 co-localized to the membrane of primary AML CD34+ blasts. Consistent with the previous studies, we also observed membrane pore formation by electron microscopy and cell necrosis by increased LDH levels rather than apoptosis(no change in Annexin V+ cell percentage and pro-apoptosis protein levels) in PNC-27 treated cells.We further evaluated the efficacy of PNC-27 in vivo . First, CD45.2 BM cells from a MLLPTD/WTFLT3ITD/ITD AML mouse model were transplanted into CD45.1 congenic recipient mice. After confirming AML development, mice were treated with PNC-27 (40mg/kg/day; n= 7) or vehicle (n=9) for two weeks. BM cells from the treated mice were transplanted into 2nd recipient mice (2×106 cells/mouse). A significantly longer survival was observed in the 2nd BM recipients from PNC-27-treated donors (n=8) compared to the 2nd BM recipients from vehicle-treated donors (n=8) (median survival: 41 days vs 32 days; p<0.01). Next, human AML cells were transplanted into NSG-SGM3 mice. Upon confirming >10% human CD45+ cell engraftment in periphery blood (PB), the mice were treated with PNC-27 (40mg/kg/day; n=10) or vehicle (n=10) for 2 weeks, and BM cells (2×106 cells/mouse) from the treated mice were transplanted into 2nd recipient mice. Significantly reduced human CD45+ engraftment in PB at 12 weeks (59.2±7.4 vs 94.6±0.94, p<0.05) and prolonged survival were observed in the 2nd BM recipients from PNC-27-treated donors (n=10) compared to the 2nd BM recipients from vehicle-treated donors (n=7). After four months follow-up, all recipients receiving BM cells from PNC-27-treated donors survived while all recipients receiving BM cells from vehicle-treated donors died (median survival was “not reached” vs 93 days, p<0.0001). Of note wild-type B6 mice treated with PNC-27 (80mg/kg) or vehicle for 2 weeks showed no difference on WBC counts and BM cell counts, suggesting no toxicity on normal hematopoiesis.In conclusion, our results suggest PNC-27 effectively targets AML LSC-enriched population by binding with cell membrane HDM2 to induce necrosis, providing a rationale for a rapid translation of this drug into the clinic. DisclosuresStein:Stemline: Consultancy; Amgen: Consultancy, Speakers Bureau.

Combinatorial Inhibition of Focal Adhesion Kinase and BCL-2 Enhances Antileukemia Activity of Venetoclax in Acute Myeloid Leukemia.

Focal adhesion kinase (FAK) promotes cancer cell growth and metastasis. We previously reported that FAK inhibition by the selective inhibitor VS-4718 exerted antileukemia activities in acute myeloid leukemia (AML). The mechanisms involved, and whether VS-4718 potentiates efficacy of other therapeutic agents, have not been investigated. Resistance to apoptosis inducted by the BCL-2 inhibitor ABT-199 (venetoclax) in AML is mediated by preexisting and ABT-199-induced overexpression of MCL-1 and BCL-XL. We observed that VS-4718 or silencing FAK with siRNA decreased MCL-1 and BCL-XL levels. Importantly, VS-4718 antagonized ABT-199-induced MCL-1 and BCL-XL. VS-4718 markedly synergized with ABT-199 to induce apoptosis in AML cells, including primary AML CD34+ cells and AML cells overexpressing MCL-1 or BCL-XL. In a patient-derived xenograft (PDX) model derived from a patient sample with NPM1/FLT3-ITD/TET2/DNMT3A/WT1 mutations and complex karyotype, VS-4718 statistically significantly reduced leukemia tissue infiltration and extended survival (72 vs. control 36 days, P = 0.0002), and only its combination with ABT-199 effectively decreased systemic leukemia tissue infiltration and circulating blasts, and prolonged survival (65.5 vs. control 36 days, P = 0.0119). Furthermore, the combination decreased NFκB signaling and induced the expression of IFN genes in vivo The combination also markedly extended survival of a second PDX model developed from an aggressive, TP53-mutated complex karyotype AML sample. The data suggest that the combined inhibition of FAK and BCL-2 enhances antileukemia activity in AML at least in part by suppressing MCL-1 and BCL-XL and that this combination may be effective in AML with TP53 and other mutations, and thus benefit patients with high-risk AML.

Elevated VMP1 expression in acute myeloid leukemia amplifies autophagy and is protective against venetoclax-induced apoptosis

Vacuole membrane protein (VMP1) is a putative autophagy protein, which together with Beclin-1 acts as a molecular switch in activating autophagy. In the present study the role of VMP1 was analysed in CD34+ cells of cord blood (CB) and primary acute myeloid leukemia (AML) cells and cell lines. An increased expression of VMP1 was observed in a subset of AML patients. Functional studies in normal CB CD34+ cells indicated that inhibiting VMP1 expression reduced autophagic-flux, coinciding with reduced expansion of hematopoietic stem and progenitor cells (HSPC), delayed differentiation, increased apoptosis and impaired in vivo engraftment. Comparable results were observed in leukemic cell lines and primary AML CD34+ cells. Ultrastructural analysis indicated that leukemic cells overexpressing VMP1 displayed a reduced number of mitochondrial structures, while the number of lysosomal degradation structures was increased. The overexpression of VMP1 did not affect cell proliferation and differentiation, but increased autophagic-flux and improved mitochondrial quality, which coincided with an increased threshold for venetoclax-induced loss of mitochondrial outer membrane permeabilization (MOMP) and apoptosis. In conclusion, our data indicate that in leukemic cells high VMP1 is involved with mitochondrial quality control.

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.

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.

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.

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.

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.

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.

Selective Anti Leukemic Activity Of Low Dose Decitabine In Combination With Ruxolitinib Against Stem/Progenitor Cells From Elderly AML Patients

Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by an accumulation of immature leukemic cells. The incidence of AML sharply increases with age. However outcomes for AML in the elderly are dismal with low response rates and high rates of relapse. Primitive AML leukemia stem cells (LSC) are resistant to conventional therapy, and potentially contribute to propensity to relapse. Therefore, new strategies to enhance targeting of AML LSCs and are required to improve outcomes. AML cells demonstrate several epigenetic alterations including enhanced promoter hypermethylation that may contribute to altered growth and self-renewal of LSC. The DNA methyltransferase (DNMT) inhibitor decitabine (DAC) has shown activity against AML LSC in preclinical studies. In clinical studies DAC is well tolerated as monotherapy and induces responses in approximately 20% of AML patients who are not candidates for conventional chemotherapy. Therefore there is considerable interest in developing combinations of DAC with other therapies for elderly patients with AML. We have previously shown that LSC from AML patients with poor risk cytogenetics demonstrate increased JAK2 tyrosine kinase activity and enhanced sensitivity to JAK2 inhibition. Since cross regulation between JAK/STAT and DNMT pathways has been reported, we evaluated the activity of the combination of DAC with the JAK2 tyrosine kinase inhibitor (TKI) Ruxolitinib (RUX) on CD34+ cells from elderly AML patients (>60 years). Exposure to low dose DAC (10 to 200nM) for 72 hours significantly inhibited the growth of primary AML CD34+ cells in a CellTitre Glo assay in a dose dependent manner (n=12, DAC 10nM 88±9% of control, 50nM 77±9%, 100nM 64±11% and 200nM 56±15%). DAC did not induce significant apoptosis in AML CD34+ cells (n=10, control, 22±5% of Annexin V positive cells vs 24±7% for the samples treated with 200nM of DAC) but dramatically and significantly reduced AML CD34+ cell growth in CFC assays (33±3%, 57±14%, 75±3% and 86±7% inhibition with 10, 50, 100 and 200nM of DAC respectively). The combination of RUX (100nM) and DAC (10 and 50nM) significantly enhanced inhibition of AML CD34+ cell growth compared to RUX or DAC alone (DAC 10nM 87±10% of control for, RUX 62±20%, DAC10+RUX 46±20%, DAC50nM 75±10%, and DAC50+RUX 43±20%, n=7, p<0.009). Combination of RUX (100nM) with 10 and 50nM DAC did not significantly enhance apoptosis in AML (n=10) or normal stem/progenitor cells (n=8, Ctrl 20.6±5%, DAC10nM 21.6±5%, RUX100nM 31.5±5%, DAC10-RUX 32.1±5%, DAC50nM 22.6±5% and DAC50-RUX 32.4±8%), but resulted in significantly enhanced inhibition of AML CD34+ CFC compared to RUX or DAC alone (n=4, CFC inhibition with DAC10 34±4%, RUX 46±15%, DAC10+RUX 68±17%, DAC50 56±15%, and DAC50+RUX 77±7%, p<0.004), but did not enhance inhibition of CB CFU-GM (n=4, CFC inhibition with DAC10 0±12%, RUX 24.3±16%, DAC10+RUX 4±9%, DAC50 9±16%, and DAC50+RUX 25±11%). These results indicate that combined targeting of both JAK/STAT and DNMT pathways can selectively inhibit the growth capacity of AML compared with normal stem/progenitor cells. We performed Western blotting on AML CD34+ cells (n=5) treated for 3 days with RUX 100nM, DAC 50nM or the combination of RUX and DAC to evaluate expression of DNMT1, DNMT3a, DNMT3b, phosphorylated STAT3 (Y705) and STAT5 (Y694) and SOCS3. Compared to the individual drugs, the combination resulted in greater reduction of p-STAT3 in 4 out 5 AML patient samples that was associated with increase in SOCS3 in 2 samples, suggesting a crosstalk between the JAK/STAT and DNMT pathways. Interestingly we did not observe enhanced inhibition of p-STAT5 with the combination. Two of five AML samples displayed decrease of DNMT1 expression and 3 of 5 samples decreased DNMT3a and DNMT3b expression upon treatment with the combination. In conclusion, our studies suggest that there may be a crosstalk between the JAK/STAT/SOCS3 signalling and DNA methylation mechanisms in CD34+ cells from elderly AML patients, and indicate that JAK2 TKI in combination with low doses of DAC selectively and significantly enhance inhibition of stem/progenitor cells from elderly AML patients compared to DNMT inhibitors alone. These results support further evaluation of this novel combination for the treatment of AML in the elderly. Disclosures:No relevant conflicts of interest to declare.