Acute Myeloid Leukemia Leukemic Stem Cells Research Articles

TIM-3/Gal-9 signaling enhances self-renewal capacity of AML-LSCs through mimicking canonical wnt signaling

Abstract (Introduction) Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs). Self-renewal capacity is one of the most important biological features of LSCs, and therefore, targeting self-renewal machineries of LSCs should be necessary for the eradication of LSCs. We have recently identified the autocrine loop consisted of LSCs-specific surface molecule TIM-3 and its ligand galectin-9 (Gal-9) in human myeloid malignancies. TIM-3/Gal-9 autocrine loop constitutively activates the nucleus accumulation of β-catenin in primary AML LSCs. However, the precise mechanism how this autocrine loop induces β-catenin accumulation has been still unknown. Here, we extended the analysis to clarify the molecular basis of the enhanced β-catenin activity in TIM-3+LSCs. (Results) We tested whether TIM-3 signaling could interact with the canonical Wnt pathway, which plays a central role in controlling the self-renewal capacity in normal hematopoietic stem cells (HSCs) through regulating intracellularβ-catenin accumulation. LDL receptor-related protein 6 (LRP6) is a key component of canonical Wnt pathway and its phosphorylation is essential for the signal transduction of Wnt signaling. Surprisingly, TIM-3 signaling induced by Gal-9 (10 ng/ml) led to phosphorylation of LRP6and accumulation of β-catenin in primary TIM-3+ AML cells. Furthermore, the phosphorylation of LRP6 induced by Gal-9 was completely abrogated in the presence of an anti-TIM-3 antibody (F38-2E2), which blocks the Gal-9 ligation to TIM-3. These results suggested that the ligation of TIM-3 by Gal-9 could activate canonical Wnt pathway independent of Wnt ligands in TIM-3+AML cells. We then tried to clarify the molecular machinery for canonical Wnt pathway activation by TIM-3/Gal-9 interaction. It has been reported that Wnt signaling promptly induces formation of the protein complex described as LRP6-signalosome preceding LRP6 phosphorylation. Therefore, we tested whether TIM-3 signaling could affect the signalosome formation. In the presence of Dickkopf-1 (Dkk-1; 200 ng/mL), which could inhibit LRP6-signalosome formation, TIM-3 signaling totally failed to phosphorylate LRP6 and to activate β-catenin accumulation, indicating that TIM-3 signaling induced Wnt signaling activation through the LRP6-signalosome formation. Since it has been shown that Src family kinases are cytoplasmic mediator of TIM-3 signaling, we focused on p120-catenin because the molecule has been identified as a substrate of Src family kinases as well as an indispensable molecule in LRP6-signalosome formation. We found that the phosphorylation of p120-catenin, a very early step of canonical Wnt signaling, was promptly induced at Tyr228 by TIM-3/Gal-9 interaction as well as Wnt ligand stimulation (Wnt3a; 200 ng/mL), leading to the enhanced β-catenin accumulation in primary TIM-3+LSCs. These results collectively suggested that the TIM-3 signaling mimicked the canonical Wnt signaling from the very early phase of its signaling cascade in LSCs. (Discussion) In this study, we showed TIM-3/Gal-9 signal mimick canonical Wnt signaling and lead to the aberrant accumulation of intracellular β-catenin in AML LSCs. Considering that TIM-3 is commonly expressed in LSCs of myeloid malignancies, but not in normal HSCs, TIM-3/Gal-9 signal can serve as a promising therapeutic target for the selective eradication of LSCs because inhibition of TIM-3 signaling can impair the self-renewal capacity of LSCs without affecting normal hematopoiesis. Disclosures Akashi: Bristol Meyers Squibb: Research Funding; Astellas Pharma: Research Funding; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd: Research Funding; Sunitomo Dainippon Pharma: Consultancy.

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.

High CD123 levels enhance proliferation in response to IL-3, but reduce chemotaxis by downregulating CXCR4 expression.

High expression of the α chain of the interleukin-3 receptor (IL-3Rα; CD123) is a hallmark of acute myeloid leukemia (AML) leukemic stem cells (LSCs). Elevated CD123 expression is part of the diagnostic immunophenotyping of myeloid leukemia, and higher expression is associated with poor prognosis. However, the biological basis of the poorer prognosis is unclear, and may include heightened IL-3 signaling and non-cell autonomous interactions with the bone marrow (BM) microenvironment. We used TF-1 cells expressing different levels of CD123 and found elevated CD123 levels amplified the proliferative response to exogenous IL-3 and maintained viability in reducing IL-3 concentrations. This was associated with stronger activation of STAT5, Akt, and extracellular signal-regulated kinase 1/2 in vitro. Surprisingly, in vivo e14.5 fetal liver cells transduced with retroviral constructs to express high CD123 failed to engraft in syngeneic recipients. In exploring the underlying mechanism for this, we found that CXCR4, a key molecule involved in LSC/BM interactions, was specifically downregulated in CD123 overexpressing cells in a manner dependent on IL-3 signaling. CXCR4 downregulation was sufficient to alter the chemotactic response of hematopoietic cells to stromal derived factor-1 (SDF-1). Thus, we propose that the overexpression of CD123 in AML LSC dictates their location by altering CXCR4/SDF-1 interaction in the BM, raising the possibility that this mechanism underpins the egress of BM AML LSC and more mature cells into the circulation.

AMPK-ULK1-Mediated Autophagy Confers Resistance to BET Inhibitor JQ1 in Acute Myeloid Leukemia Stem Cells.

Purpose: Bromodomain and extraterminal domain (BET) inhibitors are promising epigenetic agents for the treatment of various subsets of acute myeloid leukemia (AML). However, the resistance of leukemia stem cells (LSC) to BET inhibitors remains a major challenge. In this study, we evaluated the mechanisms underlying LSC resistance to the BET inhibitor JQ1.Experimental Design: We evaluated the levels of apoptosis and autophagy induced by JQ1 in LSC-like leukemia cell lines and primary CD34+CD38- leukemic blasts obtained from AML cases with normal karyotype without recurrent mutations.Results: JQ1 effectively induced apoptosis in a concentration-dependent manner in JQ1-sensitive AML cells. However, in JQ1-resistant AML LSCs, JQ1 induced little apoptosis and led to upregulation of beclin-1, increased LC3-II lipidation, formation of autophagosomes, and downregulation of p62/SQSTM1. Inhibition of autophagy by pharmacologic inhibitors or knockdown of beclin-1 using specific siRNA enhanced JQ1-induced apoptosis in resistant cells, indicating that prosurvival autophagy occurred in these cells. Independent of mTOR signaling, activation of the AMPK (pThr172)/ULK1 (pSer555) pathway was found to be associated with JQ1-induced autophagy in resistant cells. AMPK inhibition using the pharmacologic inhibitor compound C or by knockdown of AMPKα suppressed autophagy and promoted JQ1-induced apoptosis in AML LSCs.Conclusions: These findings revealed that prosurvival autophagy was one of the mechanisms involved in the resistance AML LSCs to JQ1. Targeting the AMPK/ULK1 pathway or inhibition of autophagy could be an effective therapeutic strategy for combating resistance to BET inhibitors in AML and other types of cancer. Clin Cancer Res; 23(11); 2781-94. ©2016 AACR.

Disulfiram/copper selectively eradicates AML leukemia stem cells in vitro and in vivo by simultaneous induction of ROS-JNK and inhibition of NF-\u03baB and Nrf2

Acute myeloid leukemia (AML) is a heterogeneous malignancy. Despite the advances in past decades, the clinical outcomes of AML patients remain poor. Leukemia stem cells (LSCs) is the major cause of the recurrence of AML even after aggressive treatment making, promoting development of LSC-targeted agents is an urgent clinical need. Although the antitumor activity of disulfiram (DS), an approved anti-alcoholism drug, has been demonstrated in multiple types of tumors including hematological malignancies such as AML, it remains unknown whether this agent would also be able to target cancer stem cells like LSCs. Here, we report the in vitro and in vivo activity of DS in combination with copper (Cu) against CD34+/CD38+ leukemia stem-like cells sorted from KG1α and Kasumi-1 AML cell lines, as well as primary CD34+ AML samples. DS plus Cu (DS/Cu) displayed marked inhibition of proliferation, induction of apoptosis, and suppression of colony formation in cultured AML cells while sparing the normal counterparts. DS/Cu also significantly inhibited the growth of human CD34+/CD38+ leukemic cell-derived xenografts in NOD/SCID mice. Mechanistically, DS/Cu-induced cytotoxicity was closely associated with activation of the stress-related ROS-JNK pathway as well as simultaneous inactivation of the pro-survival Nrf2 and nuclear factor-κB pathways. In summary, our findings indicate that DS/Cu selectively targets leukemia stem-like cells both in vitro and in vivo, thus suggesting a promising LSC-targeted activity of this repurposed agent for treatment of relapsed and refractory AML.

The biomarkers of leukemia stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by morphology and chromosome aberrations with high mortality. Leukemia stem cells (LSCs) in AML played important roles in leukemia initiation, progression, and were considered to be the root of chemotherapeutic drug resistance and disease relapse. The identification and targeting LSCs depended on membrane markers like CD34, CD38, CD123, TIM3, CD25, CD32 and CD96. In addition, the transcription factors were also therapeutic targets in eradicating LSCs, such as histone deacetylases (HDACs), NF-κB, HIF-1α and β-catenin. Besides membrane markers and transcription factors, intracellular reactive oxygen species (ROS), telomerase and microRNAs were identified to be new targets for ablating LSCs in AML.

Antibody-Targeted Cyclodextrin-Based Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukemia: Physicochemical Characteristics, in Vitro Mechanistic Studies, and ex Vivo Patient Derived Therapeutic Efficacy.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with high relapse rates. It is known that leukemia stem cells (LSCs), a very small subpopulation of the total number of leukemic cells, maintain the leukemia phenotype (∼80-90% of AML remain the same as at first diagnosis), display chemotherapy resistance, and contribute to disease regeneration. Therefore, targeting LSCs could control the relapse of AML. Small interfering RNA (siRNA), an effector of the RNA interference (RNAi) pathway, can selectively downregulate any gene implicated in the pathology of disease, presenting great potential for treatment of AML. In this study an antibody targeted cyclodextrin-based nanoparticle (NP) (CD.DSPE-PEG-Fab) was developed for siRNA delivery specifically to AML LSCs. The targeted CD.siRNA.DSPE-PEG-Fab formulation, where Fab specifically targets the IL-3 receptor α-chain (IL-3Rα, also known as CD123, a cell surface antigen for human AML LSCs), achieved antigen-mediated cellular uptake in KG1 cells (an AML leukemia stem and progenitor cell line). Efficient delivery of bromodomain-containing protein 4 (BRD4) siRNA using the targeted formulation resulted in downregulation of the corresponding mRNA and protein in KG1 cells and in ex vivo primary AML patient derived samples. The resulting silencing of BRD4 induced myeloid differentiation and triggered leukemia apoptosis. In addition, a synergistic therapeutic effect was detected when administered in combination with the chemotherapeutic, cytarabine (Ara-C). These results indicate the clinical potential of the antibody-tagged cyclodextrin NP for targeted delivery of therapeutic siRNA in the treatment of AML.

Evaluation of Cell Surface Markers and Targets in Leukemic Stem Cells (LSC) Reveals Distinct Expression Profiles, Unique Drug Effects, and Specific Checkpoint Regulation in AML LSC and CML LSC

In an attempt to identify novel cell surface markers and targets in leukemic stem cells (LSC) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples of patients with AML (n=257), CML (n=134), and controls (n=256: other BM neoplasms, n=116; normal/reactive BM, n=29; idiopathic cytopenia, n=31; lymphoma patients without BM involvement, n=80) for expression of cell surface markers and targets on CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells (PC) by multi-color flow cytometry. In addition, we examined mRNA expression profiles in highly purified CD34+/CD38− stem cells and CD34+/CD38+ PC by gene array- and qPCR analyses. Aberrant LSC expression profiles were identified in all patients examined. In patients with CML, CD34+/CD38− LSC expressed an almost invariable aberration profile defined as CD25+/CD26+/CD56+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably displayed aberrant surface antigens, including CD25 (55%), CD96 (35%), CD371 (CLL-1) (75%), and IL-1RAP (60%). With the exception of a subset of FLT3 ITD+ patients (45% of all FLT3-mutated cases), AML LSC did not exhibit CD26. All other markers and targets identified on AML and/or CML LSC, including CD9, CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, neural proliferation and differentiation control-1 antigen (NPDC1), and roundabout-4 (ROBO4), were also detectable on normal hematopoietic stem cells (HSC). However, several surface-targets, including CD33 and CD52, were expressed at higher levels on CD34+/CD38− LSC compared to normal HSC, and antibody-mediated targeting resulted in their selective depletion and in a significantly reduced engraftment of LSC in NSG mice. Since certain surface antigens, like CD47 (IAP), CD243 (MDR1), and CD274 (PD-L1) may contribute to intrinsic drug resistance of LSC, we also examined the expression of these antigens in AML and CML LSC. Regardless of the type of leukemia, LSC were found to express CD47 in all patients examined. MDR-1 was found to be expressed on LSC in 11/39 patients with AML (28%) and 2/22 patients with CML (9%). PD-L1 was not detectable on LSC in untreated BM samples. Exposure of LSC to interferon-gamma (IFN-G, 100 U/ml, 48 hours) resulted in expression of PD-L1 on LSC in all patients with Ph+ CML (control: 100%; IFN-G: 183±40%, p<0.05). Unexpectedly, however, IFN-G was found to induce PD-L1 expression in only 1 out of 11 AML patients tested. IFN-G showed no effects on expression of CD47 or MDR-1 in LSC. We next examined the potential mechanisms of IFN-G-mediated expression of PD-L1 on LSC and screened for drugs capable of counteracting the expression of this resistance-inducing checkpoint molecule. Of all targeted drugs examined (n=10) only the BRD4/MYC inhibitor JQ1 was identified as a regulator of IFN-G-induced PD-L1 expression on CML LSC. In particular, JQ1 (2.5 µM, 48 hours) was found to suppress IFN-G-induced (100 U/ml, 48 hours) upregulation of PD-L1 in primary CML LSC in all donors tested (IFN-G: 183±40% versus IFN-G+JQ1: 136±27% of control) suggesting that checkpoint expression in LSC is regulated by epigenetic mechanisms and MYC activity. Together, we have established cell surface marker- and target expression profiles in CD34+/CD38− AML LSC and CML LSC which should facilitate their enrichment and may support the development of LSC-eradicating treatment concepts. In addition, the unique expression profiles of LSC should provide a powerful basis for diagnostic LSC phenotyping in patients with CML and AML. DisclosuresHoermann:Novartis: Honoraria; Gilead: Research Funding; Ariad: Honoraria; Amgen: Honoraria. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Valent:Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria.

The Human Specific microRNA-941 Is a Key Member of a microRNA Signature of Functionally Validated Leukemic Stem Cells from Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is initiated and maintained by a subpopulation of leukemic stem cells (LSCs), which are defined by their property to serially engraft immunocompromised mice. Based on this a LSC-associated gene expression was identified which was shown to be prognostically relevant in a retrospective analysis (Eppert et al, Nature Medicine 2011). microRNAs (miRs) play important roles in leukemogenesis, serve as potential tumor markers and novel therapeutic targets. In the current analysis we wanted to establish a miRNA profile of functionally validated LSCs from AML patients of various molecular and cytogenetic subgroups by conducting a comprehensive functional genomics study.Method: In total, 17 de novo AML samples were sorted for CD34+ granulocyte-macrophage progenitor (GMP), CD34+ lymphoid-primed multi-potential progenitor (LMPP) and CD34- subpopulations. Sorted cells were transplanted into sublethally irradiated non-obese diabetic/severe combined immunodeficient Gamma (NSG) mice. Those subpopulations that gave rise to human leukemic cell engraftment > 1% were classified as LSCs and sub-divided into LMPP-LSCs and GMP-LSCs and compared to the CD34- cell compartment which did not give rise to human leukemic cell engraftment (non-LSC). In parallel corresponding counterparts were highly purified from healthy bone marrow (BM). miRNA libraries were prepared using the Illumina TruSeqTM Small RNA Sample Preparation Kit and sequenced as single end reads on a Illumina HiSeq 2000. The bioconductor package limma was used for identifying differentially expressed miRNAs, the made4 package for correspondence analysis and between group comparisons. Quantitative RT-PCR validation on bulk and sorted populations (LMPP-LSC, GMP-LSC, CD34-) tested the expression levels of the identified miRNAs.Results: We assessed the engraftment potential of 17 AML samples, sorted into the LMPP, GMP and CD34- fraction. The LMPP or GMP fractions of 8 patients showed a leukemic graft in the NSG mouse model, whereas the CD34- fractions were not able to show a human engraftment in these mice. To the end subpopulations of 8 validated patient samples and corresponding stem-, progenitor, and CD34- subpopulations of normal bone marrow samples (n=3) were analyzed. Global miRNA expression profiles of subpopulations from normal and patient samples were compared using correspondence analysis based between group analysis. First, LSC subpopulations were compared to the leukemic CD34- Non-LSCs: between the LMPP-LSCs and the CD34- non-LSCs 15 miRNAs were differentially expressed in the LMPP-LSCs; between the GMP-LSCs and the CD34- non-LSCs only six miRNAs were found to be differentially expressed. Using these miRNAs, an unsupervised hierarchical clustering showed that the LMPP-LSCs, the GMP-LSCs and leukemic CD34- samples formed non-overlapping clusters. In normal bone marrow, 12 miRs showed differential expression between normal BM LMPP and CD34- population and 36 miRNAs between BM GMP vs. BM CD34- cells.We then compared the miRs that were differentially expressed in the patients compared to the differentially expressed miRs between the normal BM populations: except for one miR (miR-182), the fifteen differentially expressed miRs in the LMPP-LSC versus leukemic CD34- samples did not show overlap with the twelve miR significantly differentially expressed between normal BM LMPP and CD34- cells. When the GMP-LSC to leukemic CD34- Non-LSC comparison was cross-compared to the BM GMP to normal BM CD34- comparison, only two miRs (miR-148a, miR-320a) were unique to the patient samples. When we finally compared the LMPP-LSC specific 14 miR to the 2 GMP-LSC specific miRs, interestingly, we observed that the GMP-LSC miRs were a subset of the LMPP-miRs. Thus, 14 miRs defined a set of miRs differentially expressed in functionally validated AML LSCs. Importantly, 4 of these 14 miRs represent miR-941 isoforms. This miR has not been linked to leukemogenesis yet. It is one of the very few human-specific miRs which developed de novo in the human lineage after separation of the human and the chimpanzee lineages between six and one million years ago and preferentially targets genes in hedgehog- and insulin-signalling pathways. Taken together, combining functional validation of AML LSCs with next generation sequencing allows to identify miRs so far not linked to AML biology and LSC properties. [Display omitted] DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

TIM-3/Gal-9 Signaling Enhances Self-Renewal Capacity of AML-LSCs through Mimicking Canonical Wnt Signaling

(Introduction) Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs). Self-renewal capacity is one of the most important biological features of LSCs, and therefore, targeting self-renewal machineries of LSCs should be necessary for the eradication of LSCs. We have recently identified the autocrine loop consisted of LSCs-specific surface molecule TIM-3 and its ligand galectin-9 (Gal-9) in human myeloid malignancies. TIM-3/Gal-9 autocrine loop constitutively activates the nucleus accumulation of β-catenin in primary AML LSCs. However, the precise mechanism how this autocrine loop induces β-catenin accumulation has been still unknown. Here, we extended the analysis to clarify the molecular basis of the enhanced β-catenin activity in TIM-3+LSCs.(Results) We tested whether TIM-3 signaling could interact with the canonical Wnt pathway, which plays a central role in controlling the self-renewal capacity in normal hematopoietic stem cells (HSCs) through regulating intracellularβ-catenin accumulation. LDL receptor-related protein 6 (LRP6) is a key component of canonical Wnt pathway and its phosphorylation is essential for the signal transduction of Wnt signaling. Surprisingly, TIM-3 signaling induced by Gal-9 (10 ng/ml) led to phosphorylation of LRP6and accumulation of β-catenin in primary TIM-3+ AML cells. Furthermore, the phosphorylation of LRP6 induced by Gal-9 was completely abrogated in the presence of an anti-TIM-3 antibody (F38-2E2), which blocks the Gal-9 ligation to TIM-3. These results suggested that the ligation of TIM-3 by Gal-9 could activate canonical Wnt pathway independent of Wnt ligands in TIM-3+AML cells. We then tried to clarify the molecular machinery for canonical Wnt pathway activation by TIM-3/Gal-9 interaction. It has been reported that Wnt signaling promptly induces formation of the protein complex described as LRP6-signalosome preceding LRP6 phosphorylation. Therefore, we tested whether TIM-3 signaling could affect the signalosome formation. In the presence of Dickkopf-1 (Dkk-1; 200 ng/mL), which could inhibit LRP6-signalosome formation, TIM-3 signaling totally failed to phosphorylate LRP6 and to activate β-catenin accumulation, indicating that TIM-3 signaling induced Wnt signaling activation through the LRP6-signalosome formation. Since it has been shown that Src family kinases are cytoplasmic mediator of TIM-3 signaling, we focused on p120-catenin because the molecule has been identified as a substrate of Src family kinases as well as an indispensable molecule in LRP6-signalosome formation. We found that the phosphorylation of p120-catenin, a very early step of canonical Wnt signaling, was promptly induced at Tyr228 by TIM-3/Gal-9 interaction as well as Wnt ligand stimulation (Wnt3a; 200 ng/mL), leading to the enhanced β-catenin accumulation in primary TIM-3+LSCs. These results collectively suggested that the TIM-3 signaling mimicked the canonical Wnt signaling from the very early phase of its signaling cascade in LSCs.(Discussion) In this study, we showed TIM-3/Gal-9 signal "mimick" canonical Wnt signaling and lead to the aberrant accumulation of intracellular β-catenin in AML LSCs. Considering that TIM-3 is commonly expressed in LSCs of myeloid malignancies, but not in normal HSCs, TIM-3/Gal-9 signal can serve as a promising therapeutic target for the selective eradication of LSCs because inhibition of TIM-3 signaling can impair the self-renewal capacity of LSCs without affecting normal hematopoiesis. DisclosuresAkashi:Bristol Meyers Squibb: Research Funding; Astellas Pharma: Research Funding; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd: Research Funding; Sunitomo Dainippon Pharma: Consultancy.

A High-Throughput Biology Approach to Identify Novel Therapies Specifically Targeting AML Blasts and Leukemic Stem Cells

IntroductionWhile the majority of patients diagnosed with acute myeloid leukemia (AML) will respond to standard induction chemotherapy, recurrent and refractory disease rates are high. Much needed new therapies for AML are currently in development; however, many drugs indiscriminately target rapidly proliferating cells and are ineffective against indolent, more quiescent leukemic stem cells. To identify novel therapies specific for AML blasts and leukemic stem cells (LSC), we developed a high-throughput, multi-parametric flowcytometry(FC) based method that simultaneously distinguishes specific cell populations and their differential drug responses. Using this method, we could distinguish healthy cells from leukemic stem cells and assess the effects of several drugs on these cell populations as well as the immune profile of individual patients. In addition, we identified drugs that could target both blasts and LSC and could potentially be repositioned for the treatment of AML.MethodsMononuclear cells were enriched from bone marrow (BM) aspirates collected from 10 patients by density gradient centrifugation and seeded to pre-drugged 96-well plates at 100,000 cells/well. The drugs were tested in a 10,000-fold concentration range and included standard chemotherapy agents (cytarabineandidarubicin), corticoid steroid dexamethasone,rapalogtemosirolimus, PI3K/mTORinhibitoromipalisib, JAK1/2 inhibitorruxolitinib,Srckinase inhibitordasatiniband Bcl-2 inhibitorvenetoclax. The cells were incubated with the drugs for 3 days in RPMI medium supplemented with conditioned medium from the human BM stromal cell line HS-5. The cells were stained using six different antibodies to identify blasts (CD45+, CD34+/CD33+, CD123+), LSC (CD45+, CD34+, CD38-, CD96+), healthy stem cells (HSC) (CD45+, CD34+ CD38-, CD96-), normal BM cells (CD45+, CD34-) and lymphocytes (CD45+ high, CD34-/CD33-, SSH low). We usedAnnexinV and 7-AAD to detect and remove dead and apoptotic cells. The stained cells were measured within the 96-well plates using theiQueScreener PLUS instrument (Intellicyt) in 20 minutes and at least 50,000 events acquired per well. Live cell numbers for different cell populations were calculated and dose response curves generated for each population and drug. For comparison, the viability of the samples was also assessed using theCellTiterGlo(CTG) assay.ResultsAssay set up and plate reading were accomplished within a relatively short time (staining 1 h, plate reading 20 min) with more than 5 million cell events acquired from each plate. Dose response curves generated from the live cell counts of the FC assay and viability readouts of the CTG assay were highly similar for cytotoxic drugs.(Fig. 1A).However, for targeted therapies, CTG overestimated the effect of JAK andmTORinhibitors by approximately 20% compared to the FC readouts (Fig. 1B). High variation in drug responses was observed between patient samples indicating patient and sample heterogeneity. In addition, cell populations within the same patient samples showed very different responses to the tested drugs. For example lymphocytes were sensitive tovenetoclax, but unresponsive to the other tested drugs. In contrast, blast cells were sensitive to most drugs and especially tovenetoclaxandruxolitinibwhen compared to other cell populations, suggesting that these drugs are particularly effective against this rapidly proliferating cell population.(Fig. 1C).Interestingly,ruxolitinibwas also effective at targeting LSCs, although HSCs were equally sensitive to this drug.ConclusionBy applying a high-throughput FC based method, we could easily assess the effects of several drugs on AML patient samples and distinguish the functional impact on different cell populations. The method differentiated leukemic and healthy stem cells and identified drugs that could target LSC, which are believed to be a main cause of relapses in AML. Interestingly, JAK1/2 inhibitorruxolitinib, approved for the treatment ofmyelofibrosisand polycythemiavera, was effective at targeting AML blasts and LSC. In addition, this method accounts for inter- and intra-patient variability, and may be useful for a precision medicine based approach to identify optimal, patient-specific treatment strategies. [Display omitted] DisclosuresHeckman:Celgene: Research Funding; Pfizer: Research Funding.

Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. DisclosuresNo relevant conflicts of interest to declare.

Leukemia Stem Cells Are Characterized By CLEC12A Expression and Chemotherapy Refractoriness That Can be Overcome By Targeting with Chimeric Antigen Receptor T Cells

Chemo-refractory acute myeloid leukemia (AML) is associated with poor prognosis and treatment options are extremely limited. Most of these patients are ineligible for allogeneic stem cell transplantation. Chemo-refractory AML is thought to arise due to selection pressure of resistant clones from prior use of chemotherapy or in some cases pre-exist due to properties of the leukemic stem cells (LSC). CLEC12A (also known as CLL1) has previously been described as being selectively over expressed in LSCs. Successful modalities to target CLEC12A and eradicate the LSC would overcome chemo-refractoriness in AML and would represent a vertical advance in the field.In this study, we confirm that CLEC12A is heterogenously expressed on AML blasts and over-expressed on AML LSC. We also show that CLEC12A is overexpressed on bone marrows from patients with AML that fail to achieve a complete remission after induction chemotherapy, suggesting that it could be a marker for residual disease that is refractory to chemotherapy. We then separated AML blasts into CLEC12A positive or negative cells by magnetic sorting. CLEC12A positive blasts selected from AML patients were more resistant to chemotherapy compared to CLEC12A negative blasts (20% killing of CLEC12A positive AML cells versus 43% of CLEC12A negative AML cells when cultured with cytarabine 10 µg/ml, P=0.01). This finding was confirmed by using the AML MOLM14 cell line engineered to overexpress CLEC12A. CLEC12Ahigh MOLM14 cells were more resistant to chemotherapy compared to wild type MOLM14 cells (P=0.003). We then evaluated CLEC12A resistance to chemotherapy in a patient derived AML xenograft model. We found a relative increase in CLEC12A positive cells post Ara-C induction chemotherapy in AML xenograft models (Figure 1). The observation that CLEC12A positive cells are more resistant to chemotherapy provided a solid rationale to target CLEC12A with chimeric antigen receptor T (CART) cells. We therefore developed a second generation CLEC12A directed CAR construct using CD3z and 41BB costimulatory domains and generated CLEC12A CART cells by lentiviral transduction with this construct. Upon incubation with primary AML samples or AML cell lines, CLEC12A CART cells resulted in modest effector functions, due to the heterogeneity of CLEC12A expression on AML blasts. However when CLEC12A overexpressed MOLM14 cell line or CLEC12Apos selected leukemic cells were used as targets, CLEC12A-CART cells resulted in potent cytotoxicity, proliferation and cytokine production, indicating that CLEC12A-CART cells are more specific for LSC.To test the in vivo anti-leukemic activity of CLEC12A CARTs, we used primary human AML blasts xenografted into NSG-S mice (NOD-SCID-γc-/-, additionally transgenic for human stem cell factor, IL3 and GM-CSF). Treatment with CLEC12A CART (single dose, 1x105 total T cells via tail vein injection) resulted in modest activity against AML when employed as monotherapy. To investigate the potential role of CLEC12A CART cells in eradication of MRD and LSC, mice were treated first with chemotherapy (cytarabine 60 mg/kg intraperitoneal injection daily for 5 days) followed by a single dose (1x105 total T cells via tail vein injection) of either CLEC12A CARTs or control untransduced T cells (UTD). Treatment with CLEC12A CART cells resulted in eradication of leukemia and prolonged survival in these mice (overall survival at 200 days of 100% after CLEC12A CARTs compared to 20% after UTD, p=0.01, Figure 2).In conclusion, our preclinical studies reveal that CLEC12A positive cells in leukemia are resistant to chemotherapy and can be successfully targeted with CART cells. CLEC12A CART cells can potentially be employed as a consolidation regimen after induction chemotherapy to eradicate LSC and MRD in AML. [Display omitted] [Display omitted] DisclosuresKenderian:Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Singh:Novartis: Employment. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Johnson & Johnson: Research Funding; Pfizer: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.

Aldehyde Dehydrogenase Activity in the Leukemic Stem Cell Compartment Uncovers Opposing Methylation Patterns of Leukemia Stem Cells in AML

Acute myeloid leukemia (AML) is a disease marked by abnormal differentiation of the myeloid cell lineage. Leukemia stem cells differentiate to give rise to leukemic progenitor cells (LPC) and ultimately leukemic blasts which are not leukemia-initiating. Previous studies have revealed a diverse methylation landscape in AML, but have mostly relied on the blast population rather than more purified primitive populations. Consequently, the status of the leukemic methylome during expansion from LSC to LPC and finally blast remains largely unknown. We sorted HSC from the bone marrow of normal donors and LSC, LPC, and blasts from AML patients based on expression of CD34, CD38, CD45 and aldehyde dehydrogenase (ALDH) activity. Normal HSC were defined as CD34+CD38-ALDHhigh. In AML patients, LSC were either CD34+CD38-ALDHhigh or CD34+CD38-ALDHmid; LPC were CD34+CD38+; the blast population consisted of unsorted mononuclear cells. Though patients with an ALDHhigh LSC profile may have had some residual normal HSCs present, contribution of these cells was likely minimal and thus the overall population predominantly leukemic. Methylation profiles for each cell fraction in eight untreated AML patients and five normal donors were generated using the enhanced reduced representation bisulfite sequencing (ERRBS) assay. All sequenced ERRBS libraries were aligned against the human genome (hg19) and organized into 25 base pair tiles for analysis of differentially methylated regions (DMR) using a beta-binomial model that takes variation across samples into account during DMR identification. DMR classification required a difference in methylation of >25% and false discovery rate (FDR) < 10%. Unsupervised correspondence analysis indicated that methylomes of two patients with ALDHhigh LSC were distinct from the six patients with ALDHmid LSC and therefore patients were grouped based on the ADLH activity of their LSCs for comparisons. Both ALDHhigh LSC and ALDHmid LSC had extensive alterations in methylation across their genomes when compared to HSC. The great majority of DMRs in ALDHhigh LSC were hypomethylated; of the 62,415 DMRs identified, 55,418 regions were hypomethylated while only 6,997 were hypermethylated in ALDHhigh LSC. In contrast, in ALDHmid LSC, 39,162 DMRs were hypermethylated and 5,408 regions hypomethylated compared to HSC. Despite opposing patterns of methylation, DMRs were enriched at intergenic and intragenic enhancers in both ALDHhigh and ALDHmid LSC. DMRs were functionally annotated to gene sets in the MSigDb database. Genes associated with ALDHhigh DMRs were enriched for genes with the binding motif for transcription factor Sp1 near their promoters (FDR = 2.55×10-79) and ALDHmid DMR associated genes were enriched for genes with H3K27 trimethylation in their promoters (FDR = 7.19×10-169). We compared methylation profiles of LSC to LPC and blasts in an effort to determine whether changes to the methylome occur with leukemic maturation. Interestingly, there were no significant changes in methylation between LSC and LPC in either ALDH population. However, we did see changes to the epigenome emerge when LSC or LPC were compared to leukemic blasts. In ALDHhigh patients, LSC and LPC were more hypomethylated than blasts while ALDHmid LSC and LPC were more hypermethylated than the more differentiated blasts. In conclusion, alterations to the LSC methylome were extensive and two patterns of methylation emerged based on the ALDH activity of LSC; ALDHhigh LSC displayed hypomethylated profiles and ALDHmid LSC were hypermethylated. Enrichment of DMRs at intra and intergenic enhancer regions in both LSC types despite their opposing methylation patterns highlights the importance of epigenetic marks in these regions and their role as regulators of gene expression. Significant changes in methylation between LSC or LPC and blasts, but not LSC and LPC suggest relative stability of the methylome during early leukemic differentiation with more substantial alterations occurring after the LPC level. DisclosuresGerber:Janssen: Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Carraway:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau. Gore:Celgene: Consultancy, Honoraria, Research Funding.

BCR/ABL Can Promote CD19+ Cell Growth but Not Render Them Long-Term Stemness

Cancer stem cells are a subpopulation of malignant cells that have the capacity of both self-renewal and reconstitution of the cancer. Eradication of cancer stem cells is crucial for curing the malignant disease. Previous studies in hematopoietic malignancies showed that leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) chronic phase are originated from a hematopoietic stem cell (HSC), while LSCs in acute myeloid leukemia (AML) can either be derived from HSCs or be transformed from myeloid progenitors. But in acute B-lymphoblastic leukemia (B-ALL), the origin of leukemia stem cells is not clear. In this study, we tested whether BCR/ABL could transform B-lineage committed CD19+ cells to LSCs. We found that transducing BCR/ABL in CD19+ cells can promote their colony formation in vitro and induce B-ALL like disease in vivo. However, only BCR/ABL transduced whole bone marrow cells, but not CD19+ cells, can be transplanted multiple times in recipient mice, and the frequency of long-term LSCs from the latter ranges from 1/135 to 1/629. These studies suggest that LSCs in BCR/ABL+ B-ALL may originate from CD19- hematopoietic stem/progenitor cells and that CD19 chimeric antigen receptor (CAR) modified T cell therapy may not be effective in eradicating LSCs in BCR/ABL+ B-ALL. DisclosuresNo relevant conflicts of interest to declare.

Parthenolide and DMAPT Induce Cell Death in Chronic Myeloid Leukemia Cells Via Generation of Reactive Oxygen Species

Chronic Myeloid Leukemia (CML) is a malignant hematologic disease characterized by the presence of a chromosomal alteration known as the Philadelphia Chromosome (Ph). Ph originates in Leukemia Stem Cells (LSC) and is caused by the reciprocal translocation between chromosome 9 and 22, giving rise to the Bcr-Abl oncogene, which produces a constitutively active tyrosine kinase -the BCR-ABL oncoprotein. This CML feature has made it possible the development of Tyrosine Kinase Inhibitors (TKI's) Imatinib, Dasatinib, and Nilotinib, which are able to inhibit BCR-ABL and take patients into molecular remission. However TKI's are not able to eliminate the population of LSC, which cause patients to relapse if treatment is discontinued. This has made it necessary search for alternative ways to eliminate LSC in CML patients.Parthenolide (PTL), a sesquiterpene lactone extracted from the feverfew plant (Thanacethum partenium), which has been used in folk medicine to treat inflammatory symptoms, has shown activity against LSC in Acute Myeloid Leukemia (AML). Recently Guzman and collaborators have developed an analog of PTL, named DMAPT, which is orally bioavailable. Both molecules have shown to be active inhibitors of NFkB and induce high levels of Reactive Oxygen Species (ROS) in LSC of AML. In the present study we asked whether CML cells (LSC, Progenitors and CML cell lines) are sensitive to PTL and DMAPT.Cell lines K562, Kcl-22 and HL60 (as an AML control) were cultured in liquid suspension cultures with increasing concentrations of PTL or DMAPT at a cell density of 300,000 cells per ml. At 24 hours of exposure, PTL and DMAPT showed significant induction of apoptosis at 7.5 µM and 10 µM, respectively. To determine whether NFkB activity is inhibited by PTL and DMAPT, K562, Kcl-22, HL60 cell lines, as well as CD34+ cells from a CML patient in Chronic phase where exposed to 7.5 µM PTL or 10 µM DMAPT. After 6 hours, protein lysis was induced and Western blots were performed with protein extracts from 250,000 cells per lane. Membranes where probed for Phosphorilated-p65, total p65 and b-actin as a loading control. We observed a decrease in the levels of phosphorylated p65 in cells treated with PTL and DMAPT, compared with untreated cells, while levels of total p65 remained constant.We had previosly used DCFDA stain and HMOX expression, to observe ROS induction in CML cells exposed to PTL. To asses if ROS induction is necessary for CML cell death we pretreated cells with N-acetyl cysteine (NAC), precursor of glutathione, for 1 hour before treating cells with 7.5 µM PTL or 10 µM DMAPT, and evaluated ROS levels by staining with Cell rox deep red, Mitosox-PE, and also assessing cell viability with YoPro-1 and 7AAD stain. After 6 hours of PTL treatment, we found that NAC pretreatment diminished ROS levels in cytoplasm and mitochondrial dysfunction when cells were treated with PTL. Pretreatment with NAC was able to rescue the majority of cells from apoptosis induced by PTL. Similar results were observed in cultures treated with DMAPT.Taken together, our results indicate that PTL and DMAPT were able to induce cell death via apoptosis in CML cells. This process seems to involve inhibition of p65 phosphorylation and an increase in ROS levels, which we found to be necessary for cell death to occur. DisclosuresNo relevant conflicts of interest to declare.

Development of a CD123xCD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute Myeloid Leukemia (AML)

AML is a cancer of the myeloid lineage that is characterized by the accumulation of abnormal white blood cells in the bone marrow and blood. Existing therapies do not lead to cures, partially due to their inability to eliminate residual leukemic stem cells (LSCs) in the bone marrow. T-cell redirection has been shown to be an effective method of treatment for hematologic malignancies (eg, blinatumomab) and represents an attractive approach to treat AML. CD123 (α-chain of the interleukin-3 receptor) has been shown to be expressed on the surface of AML blasts and LSCs. To eradicate CD123+ cells, we developed a bispecific antibody (JNJ-63709178) using the Genmab DuoBody® technology that can bind both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 is a humanized IgG4 bispecific antibody with silenced Fc function. This antibody is able to recruit T cells to CD123-expressing tumor cells and induce the killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50 = 0.51-0.91 nM). In contrast, this antibody does not kill CD123- cell lines, demonstrating the specificity of cytotoxicity. Consistently, the degree of cell killing correlated with the level of T-cell activation (CD69 and CD25) and cytokine release (TGF-β and TNF-α). Control bispecific antibodies containing a null arm (viral epitope) paired with a CD123 arm (CD123xnull) or a CD3 arm (nullxCD3) did not induce cytotoxicity or T-cell activation in the assays tested. JNJ-63709178 had no effect on T-cell activation when incubated with T cells alone. In AML murine xenograft models, JNJ-63709178 was able to suppress tumor growth and induce tumor regression (MOLM-13 and KG-1, respectively) in the presence of human peripheral blood mononuclear cells (PBMCs) or T cells. Tumor regression correlated with the infiltration of T cells in the tumor and the expression of T-cell activation markers such as CD25, PD1 and TIM3. Furthermore, this antibody was able to induce the killing of primary CD123+ cancer cells from the blood of patients with AML without the need to supplement with fresh T cells (EC50 = 0.83 nM). These results indicate that JNJ-63709178 can potently and specifically kill CD123+ cancer cells in vitro, in vivo and ex vivo. Pharmacokinetic studies in cynomolgus monkeys support twice weekly dosing for human studies. JNJ-63709178 is currently being investigated in a Phase 1 clinical trial in relapsed and refractory AML (ClinicalTrials.gov ID: NCT02715011). DisclosuresGaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Nemeth:Janssen Pharmaceuticals R&D: Employment, Other: stock, Patents & Royalties: patent pending. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Harman:Janssen Pharmaceuticals R&D: Employment. Millar:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Teplyakov:Janssen Pharmaceuticals R&D: Employment. Wheeler:Janssen Pharmaceuticals R&D: Employment. Luo:Janssen Pharmaceuticals R&D: Employment. Tam:Janssen Pharmaceuticals R&D: Employment, Other: stocks, Research Funding. Wu:Janssen Pharmaceuticals R&D: Employment. Chen:Janssen Pharmaceuticals R&D: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Chu:Janssen Pharmaceuticals R&D: Employment. Hughes:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Babich:Janssen: Employment. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock. Singh:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment.

Upregulation of Mir-99a Is Associated with Poor Prognosis of Acute Myeloid Leukemia and Promotes Myeloid Leukemia Cell Expansion

Acute myeloid leukemia (AML) is a hereditarily and clinically heterogeneous disease driven by a subpopulation of leukemic stem cells (LSCs) that generate the bulk of leukemic cells. LSCs can resist available treatments and result in disease progression and relapse. High number of LSCs or expression of a LSC related gene signature is independently associated with poor prognosis in AML, supporting the notion for LSCs as important targets for therapeutic intervention. It is now evident that miRNAs contribute significantly to the leukemogenic phenotype. To dissect the miRNA expression profile of LSCs, we performed miRNA array analysis in enriched LSCs cells and non-LSCs (leukemic blast cells) from bone marrow (BM) cells of the same AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a were upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients. The cohort were divided into two groups based on the median expression level of miR-99a in LSCs reference to non-LSCs, and correlation analysis indicated that higher expression levels of miR-99a in LSCs were significantly correlated with worse overall survival and event-free survival in these AML patients. Compared with cord blood CD34+ HSPCs, miR-99a was significantly highly expressed in LSCs from AML patients. The expression level of miR-99a was also higher in K562/A02 (multidrug resistant counterpart of K562 cell line) and K562/G01 (Gleevec resistant counterpart of K562 cell line) than the parental K562 cells. These results point to the importance of a novel role of miR-99a in LSC activity and disease progression.By gain-of-function studies via stable lentiviral transduction, we demonstrated that miR-99a functionally regulated the growth and survival of leukemic cells in vitro and in vivo. Enforced miR-99a expression significantly increased the clonogenic capacity in primary AML LSCs. Further studies in myeloid leukemia cell lines revealed that ectopic miR-99a led to accelerated cell-cycle progression and cell proliferation. MiR-99a overexpression (OE) also promotes leukemic cell survival after exposure to chemotherapeutic agents in AML cell lines. In vivo study in a subcutaneous xenograft model showed that a significant lower tumor inhibition rate was observed overtime in the miR-99a/OE cell injected mice compared to the Ctrl cell injected mice when treated with Ara-C. All these results demonstrated that miR-99a, the miRNA enriched from LSCs at relapse stage, plays an important role in myeloid leukemic cell survival and expansion.To explore the mechanism underlying the enhanced proliferation and chemoresistance meditated by miR-99a, we identified differential gene expression profiles by microarray analysis. The results provide a potential regulatory pathway that links the downregulated targets of miR-99a (i.e., PPP3CA) to the dysregulated key genes associated with leukemic cell expansion. Overexpression of miR-99a led to upregulation of CCNE1, and downregulation of CDKN2A which mediate G1 cell cycle arrest by stabilizing the structure of p53 or inducing p53 activation. All these results suggest an important role of miR-99a in cell cycle entry and progression, leading to the accelerated proliferation and overcoming of chemotherapeutic-agents mediated cell cycle arrest.Collectively, our study provides strong evidence that miR-99a plays an important role in altering biological properties, which may lead to the pathogenesis of myeloid leukemia and chemoresistance induction. The levels of miR-99a may be used as a biomarker for the prognosis and progression of AML. 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.

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.