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

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.

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

Blood ◽

10.1182/blood-2021-149658 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 708-708

Author(s):

Ehsan Bahrami ◽

Jan Philipp Schmid ◽

Martin Becker ◽

Anna-Katharina Wirth ◽

Rupert Öllinger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Data Analysis ◽

Therapeutic Target ◽

Leukemia Stem Cells ◽

Acute Leukemias ◽

Essential Function ◽

Pdx Models ◽

Novel Therapeutic

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.

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Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

Blood ◽

10.1182/blood.v116.21.514.514 ◽

2010 ◽

Vol 116 (21) ◽

pp. 514-514 ◽

Cited By ~ 6

Author(s):

Bin Zhang ◽

David Irvine ◽

Yin Wei Ho ◽

Silvia Buonamici ◽

Paul Manley ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Colony Formation ◽

Research Funding ◽

Leukemia Stem Cells ◽

Advisory Committees ◽

Nsg Mice ◽

Cd34 Cells ◽

Hh Signaling

Abstract Abstract 514 Background: Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo. Methods and Results: 1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone. Conclusions: We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients. Disclosures: Buonamici: Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.

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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.

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Mobilization Of Blasts and Leukemia Stem Cells by Anti-CXCR4 Antibody BMS-936564 (MDX 1338) in Patients With Relapsed/Refractory Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.3882.3882 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3882-3882 ◽

Cited By ~ 5

Author(s):

Sylvia Chien ◽

Lauren E. Beyerle ◽

Brent L. Wood ◽

Elihu H. Estey ◽

Frederick R. Appelbaum ◽

...

Keyword(s):

Clinical Trial ◽

Bone Marrow ◽

Research Funding ◽

Annexin V ◽

Fold Increase ◽

Cxcr4 Expression ◽

Leukemia Stem Cells ◽

Pre Treatment ◽

Refractory Aml ◽

Peak Value

Abstract Background Bone marrow homing of AML is dependent on CXCR4, and high levels of CXCR4 expression correlate with worse survival in AML (Rombouts et al 2004, Spoo et al 2007). CXCR4 antagonists overcome environment adhesion mediated drug resistance and enhance chemotherapy induced cytotoxicity (Liesveld et al 2007, Zeng et al 2009, Nervi et al 2009, Beider et al 2010). Plerixafor, a small molecular CXCR4 inhibitor, was studied in a phase I trial of newly diagnosed AML patients (Uy et al ASH 2011), and BMS-936564, a fully-human monoclonal antibody to CXCR4, in combination with MEC (mitoxantrone, etoposide, cytarabine), is currently under study in relapsed/refractory AML. Method The clinical trial completed a phase I dose escalation phase in AML patients, with increasing concentrations of BMS-936564 in 4 dose cohorts (0.3,1,3, and 10 mg/kg) and is currently enrolling a cohort of first salvage AML patients at the maximum dose of 10 mg/kg. The initial cohort of patients at 0.3 mg/kg received three weekly doses of antibody on days 1, 8, 15 [monotherapy period of cycle 1 (21 days)], followed by the same dose of antibody on days 1, 8, 15 of cycle 2 plus MEC chemotherapy [days 1-5 of cycle 2, (28 day cycle)]. After enrollment of the first cohort, the protocol was amended to reduce the monotherapy period to 1 week (1 dose of BMS-936564) in cohorts 1, 3, and 10 mg/kg, followed by the same combination regimen. As a companion study to this trial with the anti-CXCR4 antibody, we are investigating CXCR4 expression, timing of mobilization of leukemic blasts and leukemia stem cells (LSCs), and induction of apoptosis. Mobililzation of LSCs will be critical to eradication of leukemia, as they might serve as a reservoir for drug resistance and future relapse. We analyzed serial blast and LSC populations from blood and bone marrow samples from patients undergoing treatment by flow cytometry for phenotype, CXCR4 and annexin V expression. The putative LSCs were defined as CD34+CD38-CD123+ or by aldehyde dehydrogenase. Results An independent assessment of CXCR4 expression in 56 consecutive AML patients from our institution not related to this clinical trial revealed a mean % expression of 31%, range 1-99%, with mean fluorescence intensity (MFI) of 2092, range 319-7942. A sample of 18 patients showed a correlation in CXCR4 expression between gated blasts derived from blood and bone marrow samples from the same patient (For % expression, r2=0.85, p=5e-8; MFI r2=0.45, p=0.002). Our site has enrolled 24 AML patients thus far on the above noted trial of BMS-936564. Administration of BMS-936564 resulted in brisk mobilization of leukemic blasts in 14/24 patients that initially peaked within at 2-6 hours post start of infusion in most patients, with an average of 2.1-fold increase ± 1.8 fold (range 1.06-8.96 fold), and some blasts continued to be in circulation for days. In most cases, the samples for which mobilization was not observed either did not have circulating blasts at baseline, or were from patients who received lower doses of BMS-936564. In addition, CD34+CD38-CD123+LSCs were also mobilized post-treatment with BMS-936564, and in some cases, continued to rise over the subsequent days, during which the blast population declined. The average rise in %CXCR4 was from 29.3% pre-treatment to 69.8% peak value for blasts, and 23.0% pre-treatment to 75.6% peak value for LSCs. Although a direct correlation between CXCR4 expression by blasts and fold mobilization was not apparent, the highest fold increase in mobilization (∼9-fold) did occur in the patient with a moderately high level of CXCR4 expression, 42.5%. In this patient, there was also a sharp decline in circulating CXCR4 positive cells within 2 days and the patient achieved complete remission. BMS-936564 has demonstrated apoptosis in some preclinical models (Kuhne MR et al Clin Cancer Res v19(2): 357-66 (2013)).) For most patients, there was some increase in annexin V staining observed during the first 96 hours after antibody exposure. One patient sample with initial low baseline level of apoptosis exhibited a rise in annexin V staining (from 6% to 48%) that peaked on day 3 after administration of BMS-936564. Conclusion These data demonstrate that BMS-936564 induces mobilization of both AML blasts and LSCs, which may enhance chemotherapy-induced cytotoxicity in relapsed/refractory AML. Disclosures: Chien: Bristol-Myers Squibb: Research Funding. Cardarelli:BMS: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Becker:Bristol-Myers Squibb: Research Funding.

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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.

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Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

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mTOR Kinase Inhibitors Enhance Efficacy of TKIs in Preclinical Models of Ph-like B-ALL

Blood ◽

10.1182/blood.v128.22.2763.2763 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2763-2763 ◽

Cited By ~ 1

Author(s):

Moran Gotesman ◽

Thanh-Trang T Vo ◽

Sharmila Mallya ◽

Qi Zhang ◽

Ce Shi ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Inhibitor Therapy ◽

Cellular Viability ◽

Murine Bone Marrow ◽

Pdx Models

Abstract Background and Rationale: B-lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. While event-free survival (EFS) exceeds 85% for most patients treated with contemporary therapy, outcomes are very poor for children who relapse, highlighting a need for new treatments. In particular, children with Philadelphia chromosome-like (Ph-like) B-ALL (who lack BCR-ABL1 rearrangement) have high rates of relapse and mortality with conventional chemotherapy. Transcriptional profiling and genomic sequencing of Ph-like ALL specimens have identified a variety of alterations that activate oncogenic kinase signaling, including rearrangements (R) of CRLF2, ABL1, and PDGFRB. Addition of the tyrosine kinase inhibitor (TKI) imatinib to chemotherapy has dramatically improved EFS for patients with BCR-ABL1-rearranged (Ph+) B-ALL, and it is hypothesized that TKI addition to therapy will similarly improve outcomes for patients with Ph-like ALL. Our prior preclinical studies in Ph+ B-ALL demonstrated enhanced efficacy of combining TKIs (imatinib or dasatinib) with mTOR kinase inhibitors (TOR-KIs) (Janes et al., Nature Medicine 2010; Janes et al, Leukemia2013). In the current studies, we hypothesized that dual kinase inhibitor therapy would have superior anti-leukemia cytotoxicity in Ph-like ALL and thus investigated combined TKI and TOR-KI treatment using patient-derived xenograft (PDX) models of childhood Ph-like ALL. Methods: For in vitro studies, viably cryopreserved leukemia cells from established ABL1-R Ph-like ALL PDX models (2 ETV6-ABL1) were incubated with the TKI dasatinib, TOR-KIs, or both TKI + TOR-KI for 72 hours prior to flow cytometric assessment of cellular viability via Annexin V and propidium iodide staining. Two chemically distinct TOR-KIs (MLN0128 or AZD2014) were used to confirm on-target effects. Additional primary ABL1-R or PDGFRB-R Ph-like ALL specimens were plated in methylcellulose without or with inhibitors in colony-forming assays. Phosphoflow cytometry (PFC) analysis of ALL cells incubated with inhibitors was also performed to measure the ability of TKIs and TOR-KIs to inhibit intracellular ABL1 and PI3K/mTOR signaling pathways. For in vivo studies, Ph-like ALL PDX models were treated with dasatinib, the TOR-KI AZD8055, or both drugs via daily oral gavage for 8 days. Human CD19+ ALL was quantified in murine spleens and bone marrow at end of treatment with quantification of cycling cells by EdU incorporation. PFC analysis of murine bone marrow was also performed 2 hours after drugs were dosed, to measure in vivo inhibition of signaling proteins. Results: Combined in vitro treatment with dasatinib and MLN0128 or AZD2014 decreased cellular viability more than inhibitor monotherapy. Similarly, in a set of CRLF2-rearranged samples, mTOR inhibitors augmented killing by the JAK2 inhibitor BBT-594. Incubation of primary ABL1-R or PDGFRB-R ALL cells with both dasatinib and AZD2014 more robustly inhibited colony formation than did inhibitor monotherapy. In in vitro PFC analyses of ABL1-R samples, we observed expected dasatinib-induced inhibition of phosphorylated (p) STAT5. Inhibition of the mTOR substrate pS6 was observed with dasatinib, MLN0128, and AZD2014 with more complete inhibition achieved when dasatinib combined with either MLN0128 or AZD2014. Similarly, in vivo treatment of PDX models with dasatinib and AZD8055 reduced leukemia burden and pS6 signaling more completely than either inhibitor alone. Importantly, dual inhibition decreased the percentage of cycling human ALL cells in murine bone marrow, but preserved cycling in normal mouse bone marrow cells in the same animals. Our data thus provide additional compelling preclinical rationale for combined inhibitor therapy with TKIs and TOR-KIs in Ph-like ALL. Disclosures Weinstock: Novartis: Consultancy, Research Funding. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

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Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-151671 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1170-1170

Author(s):

Anilkumar Gopalakrishnapillai ◽

Anne Kisielewski ◽

Yang Zhang ◽

Bruce Ruggeri ◽

Peggy Scherle ◽

...

Keyword(s):

Median Survival ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Research Funding ◽

Publicly Traded ◽

Pediatric Acute Myeloid Leukemia ◽

Patient Derived Xenograft ◽

Pediatric Aml ◽

Pdx Models ◽

Acute Myeloid

Abstract Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML. We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints. Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

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Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v116.21.778.778 ◽

2010 ◽

Vol 116 (21) ◽

pp. 778-778

Author(s):

Zhihong Zeng ◽

Yuexi Shi ◽

Twee Tsao ◽

Yihua Qiu ◽

Steven M. Kornblau ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Signaling Pathways ◽

Research Funding ◽

Kinase Inhibitor ◽

Annexin V ◽

Mtor Signaling ◽

Leukemic Cells ◽

Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

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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.

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