In Vivo CRISPR-Cas9 Screens in PDX Models Reveals ADAM10 As Novel Therapeutic Target in Acute Leukemia

Abstract Acute leukemias require more accurate and effective treatments, especially upon disease relapse. In search for novel therapeutic targets for acute leukemias, we established a pipeline for CRISPR-Cas9 mediated functional genomic screens which harbor the ability to elegantly increase our knowledge about vulnerabilities and gene dependencies. For a highly patient-related setting, we performed CRISPR knockout (KO) dropout screens in patient-derived xenograft (PDX) models in vivo, combining the advantages of studying an individual patient´s tumor cell in the physiologic in vivo bone marrow microenvironment. Serially transplantable PDX models were lentivirally transduced to stably express Cas9. A customized CRISPR-Cas9 library targeting about 100 genes addressing surface molecules was designed, cloned and transduced into two PDX models of acute lymphoblastic leukemia (ALL). Enriched PDX ALL cells were transplanted into NSG mice and grown until advanced disease stage. Input versus end stage cells were subjected to next generation sequencing, followed by data analysis using MAGeCK algorithm. Data analysis revealed commonly depleted as well as sample-specific depleted genes between the two PDX models tested, and CXCR4 and ITGB1 were the top commonly depleted genes from the screens. For target validation, single sgRNA were cloned into the knockout vector; concomitant expression of recombinant fluorochromes allowed competitive growth assays in PDX models in vivo, comparing cells with KO of interest versus control KO in the same animal. In vivo competitive assay showed that both PDX models clearly depended on both CXCR4 and ITGB1, validating an essential function for both genes in the two PDX ALL models. Of note, disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was among the list of dropout genes in the screens. ADAM10 is known for its role in the central nervous system and considered as a therapeutic target in Alzheimer disease, but poorly studied in the context of leukemia. In competitive validation assays in vivo, ADAM10 KO population showed a clear growth disadvantage compared to control KO cells in a number of PDX models of ALL, but also acute myeloid leukemia (AML); in some PDX models, ADAM10 KO cells were completely abrogated, indicating that ADAM10 plays an essential role for different types of acute leukemias and represents a yet unknown vulnerability. To better characterize the role of ADAM10 for the clinical situation, we performed further in vivo assays with PDX models. Re-expression of ADAM10 in ADAM10 KO PDX cells could partially rescue the phenotype in an in vivo competitive reconstitution assay, unequivocally proving ADAM10 essentiality in ALL cells. Interestingly, a similar rescue assay expressing a ADAM10 variant lacking the disintegrin domain resulted in the same phenotypical compensation, highlighting essentiality of the enzymatic but not adhesion domain of ADAM10 in tumor engraftment and growth in BM. Important for translating the molecular insights into clinical use, PDX cells treated with an ADAM10 chemical inhibitor ex vivo, showed reduced tumor engraftment capacity compared to the vehicle treated cells, suggesting a role for ADAM10 in tumor-niche interactions and homing to the bone marrow. Further, we performed limiting dilution transplantation assays to determine stem cell frequencies; ADAM10 loss resulted in reduced stemness and a reduced number of leukemia-initiating cells compared to control KO cells, indicating that ADAM10 is essential also in leukemia stem cells. Of clinical relevance, ADAM10 KO significantly sensitized leukemic cells towards treatment of mice with the routine chemotherapeutic drug Cyclophosphamide in vivo, suggesting a putative synergistic effect when addressing ADAM10 as a therapeutic target. In summary, our data revealed ADAM10 as attractive novel vulnerability in acute leukemias with essential function for the tumor-niche interaction, leukemia stem cells and anti-leukemia treatment. ADAM10 might be addressed as therapeutic target to treat acute leukemias in the future. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Imetelstat Significantly Reduces Leukemia Stem Cells in Patient-Derived Xenograft Models of Pediatric AML

Blood ◽

10.1182/blood-2021-151607 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3352-3352

Author(s):

Sonali P. Barwe ◽

Fei Huang ◽

E. Anders Kolb ◽

Anilkumar Gopalakrishnapillai

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Research Funding ◽

Annexin V ◽

Telomerase Activity ◽

Leukemia Stem Cells ◽

Patient Derived Xenograft ◽

Pediatric Aml ◽

Pdx Models

Abstract Introduction Acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite the use of maximally intensive therapy, 20% of patients experience recurrent disease. These patients are also burdened with significant treatment-related toxicities. To improve survival in pediatric AML, novel targeted therapies that are more effective and less toxic are needed. Telomerase inhibition has been shown to be effective in reducing leukemic burden and eradicating leukemia stem cells (LSCs) in syngeneic mouse models of AML and in patient-derived xenograft (PDX) models of adult AML (Bruedigam et al., 2014). Recent transcriptome analyses demonstrate that the genomic landscape of pediatric AML is distinct from adult AML (Bolouri et al., 2018). In fact, mutations in the telomerase complex components are infrequent in pediatric AML unlike adult AML patients (Aalbers et al., 2013). However, similar to what is seen in adult patients, Aalbers et al. identified that telomere lengths in pediatric AML cells were shortened compared to normal leukocytes, and pediatric AML patients with the shortest telomere length tend to have shorter overall survival. Furthermore, the 5-year survival rate was 88% for pediatric AML patients who had lower telomerase activity, and 43% for those patients with higher telomerase activity, suggesting telomerase activity could be an important prognostic factor in pediatric AML patients (Verstovsek et al., 2003). Imetelstat is an oligonucleotide that specifically binds with high affinity to the RNA template of telomerase and is a potent, competitive inhibitor of telomerase enzymatic activity (Asai et al., 2003; Herbert et al., 2005). In this study, we evaluated if imetelstat has anti-leukemia activity in pediatric AML PDX models. Results The PDX lines tested in this study were derived using samples from pediatric AML patients who were 1-14 years old, representing different FAB subtypes. Mouse passaged pediatric AML PDX lines (n=6) were treated ex vivo with imetelstat or mismatch oligo control and the viability of LSC (CD34+CD38low population) was determined at 48 or 96 h by staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Imetelstat treatment significantly increased apoptosis/death (PI+/annexin V+) of the LSC population in a dose-dependent manner in all PDX lines evaluated (Fig. 1A, B), while it had limited activity on LSCs in normal pediatric bone marrow samples (n=4). The efficacy of imetelstat either alone or in combination with chemotherapy or azacitidine was evaluated in two distinct PDX models of pediatric AML in vivo. Mice engrafted with both NTPL-377 and DF-2 lived longer when treated with imetelstat than the untreated mice (Fig. 1C, D, n=5 each, P<0.05). Mice receiving standard chemotherapy consisting of cytarabine and daunorubicin or azacitidine showed prolonged survival compared to the untreated mice. Interestingly, sequential administration of imetelstat following chemotherapy treatment provided additional benefit over chemotherapy alone (P<0.01). Concurrent treatment of azacitidine and imetelstat further extended survival of these mice compared to azacitidine alone (P<0.05). At the end of the in vivo studies, the percentage of LSC population was evaluated in the bone marrow of mice post euthanasia. There was a significant reduction of LSC population in mice treated with imetelstat compared to those treated with the mismatch oligo (Fig. 1E, F, P<0.05). Neither chemotherapy nor azacitidine alone affected LSC population compared to untreated mice. However, imetelstat significantly reduced the LSC population when combined with chemotherapy or azacitidine compared to single agent (P<0.05). These results were confirmed by secondary transplantation in mice, which showed delayed engraftment of cells isolated from imetelstat treated mice (Fig. 1G, H). Conclusions Imetelstat treatment of pediatric AML PDX samples showed significant dose- and time-dependent effects on the viability of the LSCs to induce cell apoptosis/death. These results were corroborated in vivo in two distinct PDX models which showed reduced LSC population and increased median survival in mice with imetelstat treatment. Combining imetelstat with chemotherapy or azacitidine further enhanced activity against LSCs, suggesting imetelstat could represent an effective therapeutic strategy for pediatric AML. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Huang: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Gopalakrishnapillai: Geron: Research Funding.

Download Full-text

Treatment with 8F4, An Anti-PR1/HLA-A2 T Cell Receptor-Like Antibody, Reduces Established Acute Myeloid Leukemia and Eliminates Leukemia Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.766.766 ◽

2011 ◽

Vol 118 (21) ◽

pp. 766-766

Author(s):

Anna Sergeeva ◽

Hong He ◽

Kathryn Ruisaard ◽

Karen Clise-Dwyer ◽

Lisa S St. John ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Cell Receptor ◽

Leukemia Stem Cells ◽

Nsg Mice ◽

Secondary Transfer

Abstract Abstract 766 PR1 (VLQELNVTV) is an HLA-A2-restricted leukemia-associated peptide from proteinase 3 and neutrophil elastase that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of myeloid leukemia. We developed a high affinity T cell receptor (TCR)-like mouse monoclonal antibody (8F4) that binds to a conformational epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes. Moreover, 8F4 mediated complement dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSC), but not normal leukocytes. To investigate in vivo biological effects 8F4 on established leukemia, we established xenografts of primary human HLA-A2-positive AML in sublethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Leukemia engraftment was monitored in peripheral blood by flow cytometry. Mice with established PR1/HLA-A2-expressing leukemia were treated with twice-weekly intravenous injections of 200 μg 8F4 or isotype control antibody. Flow cytometry and histology analysis of tissues was used to assess leukemia burden and level of engraftment. After 5 weeks of treatment AML was reduced 300-fold in bone marrow of 8F4-treated mice compared to isotype-treated control animals (0.07 ± 0.06% hCD45+cells versus 20.4 ± 4.1%, n=5 mice per group). Moreover, leukemia stem cells (LSC, CD34+CD38−Lin-) were no longer detected in bone marrow of 8F4-treated mice, compared to 0.88 ± 0.24% in isotype-treated mice. Equally, AML was evident in the liver and spleen of isotype-treated mice (1.1 ± 0.16% and 0.32 ± 0.17%, respectively), but was undetectable in 8F4-treated mice (p<0.001). Similar results were obtained with AML from two additional patients, one with secondary AML (CMML) and one with AML-M7. Bone marrow contained 6.2 ± 3.0% (n=3) AML versus 41 ± 15% (n=2 mice; p=0.06) in the first case and 0.16 (n=1) versus 7.0 ± 4.1 (n=2) in the second case after 2–3 weeks of twice-weekly injection. To confirm 8F4-mediated elimination of LSC, we performed secondary transfer experiment with 1×106 bone marrow cells from 8F4- and isotype-treated mice, transplanted into recipient NSG mice, irradiated with 250 cGy. AML was undetectable in mice that received bone marrow from 8F4-treated animals versus 4.1 ± 2.4% (n=4) in bone marrow of mice that received cells from isotype- treated mice, determined at 16 weeks after secondary transfer. Because PR1/HLA-A2 expression on normal hematopoietic cells (HSC) is similar to LSC in AML patients, we sought to determine whether 8F4 treatment of NSG mice xenografted with CD34-selected umbilical cord blood resulted in elimination of xenograft. Fourteen weeks after transplant stable chimerism (4.1 - 7.7% hCD45+ cells) was established, mice were treated with 50 μg 8F4 intravenously and peripheral blood was monitored weekly for chimerism. Human CD45+ cells decreased to 0.35 – 0.95% by week 1, but increased to 1.9 – 2.1 % hCD45+ cells at week 3. Bone marrow at week three contained myeloid (CD13+CD33+) and lymphoid (CD19+) cells showing that while 8F4 has off- target effects against normal hematopoietic cells, HSC are preserved. This is consistent with our previous studies that showed no 8F4-mediated effect on colony formation of normal bone marrow cells. In conclusion, these results show that anti-PR1/HLA-A2 monoclonal antibody 8F4 is biologically active in vivo and selectively eliminates LSC, but not normal HSC. This justifies continued study of 8F4 as a novel therapy for AML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Epigenetic Profiling Of Leukemia Stem Cells In a Model Of TET2/FLT3-Mutant AML

Blood ◽

10.1182/blood.v122.21.476.476 ◽

2013 ◽

Vol 122 (21) ◽

pp. 476-476

Author(s):

Alan H. Shih ◽

Yanwen Jiang ◽

Kaitlyn Shank ◽

Suveg Pandey ◽

Agnes Viale ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Targeted Therapies ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Leukemia Stem Cells ◽

Self Renewal ◽

In Vivo Models ◽

Flow Cytometric

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj <0.05). Of note, we found that genes involved in normal myelo-erythroid differentiation, including GATA1, GATA2, and EPOR, were transcriptionally silenced in LSCs relative to normal stem cells, consistent with their the impaired differentiation and increased self-renewal observed in LSCs. Enhanced representation bisulfite sequencing revealed a subset of these genes were marked by increased promoter methylation. The number of hyper differentially methylated regions (HyperDMRs, 10% methylation difference, FDR<0.2) was significantly greater in Vav+Tet2fl/flFlt3-ITD cells (787 HyperDMRs) compared to Vav+Tet2fl/fl cells (76 DMRs) suggesting FLT3 activation and TET2 loss cooperate to alter the epigenetic landscape in hematopoietic cells. Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Targeting Myeloma Cells and Their Progenitors By Pim Kinase Inhibition

Blood ◽

10.1182/blood.v124.21.5724.5724 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5724-5724

Author(s):

Shiro Fujii ◽

Hirokazu Miki ◽

Asuka Oda ◽

Keiichiro Watanabe ◽

Ryota Amachi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Drug Resistance ◽

Abc Transporter ◽

Therapeutic Target ◽

Bone Lesions ◽

Drug Resistant ◽

Acidic Conditions ◽

Important Therapeutic Target

Abstract Myeloma (MM) cells grow and expand almost exclusively in the bone marrow while creating a cellular microenvironment suitable for MM cell growth and survival (MM niche). In pursuing the molecular mechanisms whereby MM cells gain drug resistance in the “MM niche”, we have found that the serine/threonine kinase Pim-2 is constitutively over-expressed in MM cells, and further up-regulated by co-cultures with bone marrow stromal cells (BMSCs) as well as osteoclasts (Leukemia, 2011), and that Pim-2 is an important therapeutic target in MM for the progression of MM tumor and bone disease (Leukemia, 2014). The ABC transporter BCRP is preferentially expressed in drug resistant MM cells as well as in MM progenitors or stem cells. BCRP has been demonstrated to be phosphorylated by Pim kinases to trigger its dimerization and function; Pim inhibition may suppress the BCRP function to sensitize BCRP-expressing MM cells to chemotherapeutic agents. In the present study we therefore explored whether Pim inhibition is able to target and impair BCRP-expressing drug-resistant MM cells and MM progenitors. We analyzed an ABC transporter activity in BCRP-expressing RPMI8226 and KMS11 cells by intracellular accumulation and retention of BCRP substrates with auto-fluorescence emission, mitoxantrone and doxorubicin, in flow cytometry. Treatment with Pim inhibitors, SMI-16a or SMI-4a, increased the incorporation of these drugs into the MM cells and enhanced their subsequent intracellular retention after 6-hour incubation without these drugs, although BCRP expression on their surface was only marginally affected by the Pim inhibition. Interestingly, acidic conditions up-regulated Pim-2 expression while reducing the accumulation and retention of these drugs in BCRP-expressing RPMI8226 and KMS11 cells. However, the Pim inhibitors efficaciously restored the drug accumulation and retention reduced by extracellular acidification, and enhanced the cytotoxic activity of the BCRP substrate doxorubicin against RPMI8226 cells rather preferentially in acidic conditions. Furthermore, the Pim inhibition minimized the sizes of “side populations”, highly drug-resistant fractions with enhanced BCRP activity, and the ability of colony formation in RPMI8226 and KMS11 cells, which was more marked in acidic conditions. We previously demonstrated the in vivo effects of the Pim inhibitors in human INA-6 cell-bearing SCID-rab MM models and syngeneic mouse MM models with an intra-tibial inoculation of 5TGM1 MM cells (Leukemia, 2014). To further examine the acid-tropism of anti-tumorigenic activity of Pim inhibition, we pretreated murine 5TGM1 MM cells in vitro with or without SMI16a at pH6.8 for 24 hours, and transplanted to the tibiae in mice the same numbers of viable MM cells remaining in each treatment group. Treatment with SMI16a at pH6.8 almost completely abrogated in vivo tumorigenic capacity of 5TGM1 cells, while MM cells without the treatment rapidly grew and expanded in and outside of the tibiae, suggesting targeting clonogenic MM cells by Pim inhibition preferentially in acidic conditions. Taken together, Pim-2 may become an important therapeutic target of drug-resistant BCRP-expressing MM cells and their progenitors which appear to gain more drug resistance in acidic bone lesions. Combinatory treatment with Pim inhibitors warrants further study to overcome drug resistance in MM cells, including their tumorigenic cancer stem cells. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells

Blood ◽

10.1182/blood.v118.21.553.553 ◽

2011 ◽

Vol 118 (21) ◽

pp. 553-553

Author(s):

John M Ashton ◽

Marlene Balys ◽

Sarah Neering ◽

Glenn Cowley ◽

David E. Root ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Leukemic Cell ◽

Gene Signature ◽

Leukemic Cells ◽

Leukemia Stem Cells ◽

Human Leukemia ◽

Normal Cells ◽

In Vivo Growth

Abstract Abstract 553 In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Targeting F2r/Beta-Catenin/JNK Signaling for Leukemia Stem Cell Eradication

Blood ◽

10.1182/blood.v126.23.3061.3061 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3061-3061

Author(s):

Jennifer Lynch ◽

Hangyu Yi ◽

Brendon Martinez ◽

Florida Voli ◽

Jenny Yingzi Wang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Death ◽

Blood Cells ◽

Mitochondrial Membrane ◽

Leukemia Stem Cells ◽

Ros Production ◽

Self Renewal ◽

Jnk Signaling

Abstract G-protein coupled receptors (GPCRs) are the most successful drug targets with 36% of currently marketed drugs targeting human GPCRs. F2r, a GPCR that is overexpressed in various human cancers (Ribeiro et al., Oncol Rep 2009), is a positive regulator of the β-catenin pathway and an inhibitor of the JNK pathway in mammalian cells (Sun et al., Nat Cell Biol 2001). The expression of F2r is significantly elevated in aggressive leukemias including blast phase of CML and AML (Veiga et al., Blood Cells Mol Dis 2011). While these observations implicate an involvement of F2r in human leukemia, its function in AML remains unknown. Our preliminary data showed that shRNA-mediated knockdown of F2r markedly decreased active β-catenin in MLL-AF9 mediated pre-leukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs), confirming that F2r is a positive regulator of β-catenin. F2r deficiency decreased LSC colony formation (p=0.0002) in a serial replating assay, indicating a reduction in self-renewal capacity. Furthermore, LSCs exhibited a significant enhancement in apoptotic activity in response to F2r deficiency, displaying a 12-fold increase in apoptosis. Our microarray expression analysis revealed that F2r inhibition significantly reduced the expression of several genes responsible for maintenance of mitochondrial integrity and energy metabolism (mtND4L (p=0.0013), mtND2 (p<0.0001) and mtCytB (p<0.0001)). To investigate this further we used a mitochondria-specific fluorogenic probe to measure reactive oxygen species (ROS) production. A significant increase in ROS production (p=0.0003) indicated that F2r inhibition destabilizes the mitochondrial membrane. This was accompanied by a marked increase, as observed by Western blot analysis, in the proapoptotic proteins Bcl-2-interacting mediator of cell death (Bim) and thioredoxin-interacting protein (Txnip) which permeabilize the mitochondrial membrane releasing cytochrome c and inducing apoptosis. Through oxidative phosphorylation, mitochondria play an essential role in the supply of metabolic energy (ATP) to the cell. F2r deficient LSCs had a significantly reduced rate of oxygen consumption measured using a phosphorescent oxygen probe (p=0.0066) and a significantly lower concentration of basal ATP (p=0.012) compared to control LSCs. F2r inhibition, therefore, induces substantial oxidative stress which triggers the intrinsic apoptotic pathway. To assess the therapeutic value of F2r inhibition, we used the selective non-peptide F2r inhibitor SCH79797. Alamar blue-based cell viability assays showed that SCH79797 was potent against LSCs and had no cytotoxic effects on lineage-negative normal mouse bone marrow cells. F2r inhibitor treatment resulted in a 2-fold reduction in colony forming ability, 3.8-fold enhanced ROS production and inhibited β-catenin activity. BrdU labeling revealed a significant reduction in in vivo short-term proliferation of LSCs that were pre-treated with SCH79797 for 48 hours in culture, transplanted into recipient mice and collected from bone marrow 8 days post-transplantation (p=0.0008). Additional in vivo studies using a mouse model of MLL AML are currently ongoing to further evaluate the therapeutic potential of F2r inhibition. Collectively, our findings suggest that F2r inhibition selectively targets LSC self-renewal, identifying a therapeutic window to eliminate LSCs while preserving normal blood cells. Previous studies suggest that F2r knockdown not only suppresses β-catenin but also activates JNK signaling. Consistently, our Western blot analysis revealed activation of JNK in response to inhibition of F2r. Sustained JNK activation has been reported in many types of AML cells and promotes survival signals during leukemia development (Hess et al., Nat Genet 2002). This suggests that JNK and F2r inhibition could be used in combination to impair LSC self-renewal, with a concurrent increase in cell death. In support of this hypothesis, we have showed that co-inhibition of F2r and JNK induced a potent anti-LSC effect, significantly increasing cell death and ROS production compared to single treatment. The efficacy of this co-treatment is currently being evaluated in primary human AML patient samples in addition to our in vivo mouse model system. Altogether our data suggest a novel LSC-eliminating treatment strategy targeting F2r/β-catenin/JNK signaling for aggressive AML. Disclosures No relevant conflicts of interest to declare.

Download Full-text

In vivo targeting of leukemia stem cells by directing parthenolide-loaded nanoparticles to the bone marrow niche

Leukemia ◽

10.1038/leu.2015.343 ◽

2015 ◽

Vol 30 (7) ◽

pp. 1582-1586 ◽

Cited By ~ 24

Author(s):

H Zong ◽

S Sen ◽

G Zhang ◽

C Mu ◽

Z F Albayati ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Leukemia Stem Cells ◽

Bone Marrow Niche

Download Full-text

Leukemia Stem Cells Are Resistant to In Vivo, Cell Non-Autonomous Wnt Inhibition.

Blood ◽

10.1182/blood.v114.22.1025.1025 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1025-1025

Author(s):

Steven W. Lane ◽

Cristina Lo Celso ◽

Stephen M Sykes ◽

Sebastian Shterental ◽

Mahnaz Paktinat ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Wnt Signaling ◽

Bone Marrow Microenvironment ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Dkk1 Expression ◽

Hsc Niche

Abstract Abstract 1025 Poster Board I-47 Acute myeloid leukemia (AML) initiating cells reside within and utilize the bone marrow microenvironment, as a sanctuary to evade chemotherapy and to maintain self-renewal. Following treatment, these leukemia stem cells (LSC) re-emerge and reconstitute disease, leading to relapse. The canonical Wnt signaling pathway is frequently dysregulated in LSC and recent data indicates that Dkk1 (a potent endogenous Wnt inhibitor) may have a therapeutic role in treating AML. Microenvironment specific Dkk1 expression inhibits hematopoietic stem cell (HSC) Wnt and extinguishes HSC self-renewal in vivo, identifying the Wnt pathway as essential in normal HSC-niche homeostasis. We investigated the importance of bone marrow microenvironment Wnt signaling in LSC survival. AML was generated using retroviral transduction of murine bone marrow with the MLL-AF9 fusion oncogene. We then assessed the potential for niche-directed Wnt inhibition of LSC using 2.3kbColl1alpha-Dkk1 transgenic mice in which Dkk1 expression is restricted to osteoblasts. AML was observed in the Dkk1 or wild type mice with similar disease latency and phenotype. AML was also observed in secondary transplant recipients, although there was a reduction of LSC (linlowcKithighSca-1-FcGRII/III+CD34+) derived from Dkk1 mice (LSC frequency 2.8% WT vs 1.6% Dkk1, p<0.05), correlating with a subtle prolongation in disease latency (n=15, 20 days WT vs. 24 days Dkk1, p<0.001). To determine the status of Wnt signaling in MLL-AF9 AML, we generated AML in bone marrow derived from TOPGal reporter mice that harbor a Tcf/Lef responsive promoter with a LacZ reporter, and quantified LacZ expression or galactosidase protein levels. Wnt activation was increased following transformation of bone marrow with MLL-AF9 (relative TOPGal expression 1.35 empty vector vs 2.58 MLLAF9, p=0.03). To assess the effects of osteoblast-restricted Dkk1 expression in vivo, Wnt signaling was measured in LSC purified by high-speed multiparameter flow cytometry. Reporter activity (fluorescein di-β-D-galactopyranoside (FDG), Invitrogen) was unchanged in LSC from WT or Dkk1 recipients (Median fluorescent intensity 552 vs 542, p=0.85), indicating that, in contrast with normal HSC, Wnt signaling in LSC is relatively resistant to Dkk1 expression in the niche. To better understand the mechanism of LSC resistance to Dkk1, we examined the homing and micro-localization of LSC in vivo using live, 3 dimensional two photon-confocal hybrid imaging of the bone marrow microenvironment. LSC proliferate with similar kinetics in Dkk1 or WT recipients (proliferating fraction 57.7% WT vs 50.3% Dkk1 LSC p=0.48). However, when compared to HSC, LSC home with less affinity to osteoblasts and may escape the effects of osteoblast specified Dkk1 expression through residence in a niche that is physically distant from endosteum (Median distance to osteoblast 18um WT vs 20.6um Dkk1 LSC, p=0.13). Taken together, these data indicate that MLL-AF9 LSC can escape the normal HSC-niche homeostatic constraints regulated by Wnt, an observation that may have important therapeutic implications. Disclosures: Scadden: Fate Therapeutics: Consultancy. Gilliland:Merck: Employment.

Download Full-text