Resistant Leukemia Stem Cells Research Articles

High Throughput Drug Screening of Leukemia Stem Cells Reveals Resistance to Standard Therapies and Sensitivity to Other Agents in Acute Myeloid Leukemia

Background:Leukemia stem cells (LSCs) play a critical role in AML propagation and relapse. Other investigators have also highlighted unique gene expression profiles for the leukemia stem cell population. Here we compared the results of in vitro drug sensitivity testing against a custom panel of drugs and drug combinations for blast populations vs. leukemia stem cell populations derived from the same patients, as well as mutation analysis for a panel of 194 recurrently mutated genes in AML.Patients and Methods:Patient AML samples were obtained with IRB approval. LSCs were isolated by fluorescence-activated cell sorting (FACS) and the blast population enriched to >90% using immunomagnetic beads from blood samples from 5 patients with AML. A sixth AML patient sample was used for NOD/SCID IL2R γc−/− engraftment, in order to compare characteristics of pre- and post-engraftment subclones. Our CLIA approved custom assay includes 153 drugs and targeted agents, both FDA approved and investigational, with additional drug combinations. High throughput screens (HTS) were conducted with enriched cells adherent to matrix protein in 384 well plates with 8 concentrations of each drug spanning 4 logs. Viability was assessed with CellTiter-Glo (Promega). HTS were performed on LSCs, blasts and pre- as well as post-engraftment AML subclones from the xenograft. Dose-response curves were generated to calibrate drug resistance patterns. Mutation analysis by NGS for a panel of 194 recurrently mutated genes in AML (MyAML®) including 37 translocations was also conducted for the LSC and blast populations.Results:AML blasts and LSCs exhibited divergent drug susceptibility patterns (see volcano plot in Figure). Of 11 drugs commonly used in AML, 8 were typical chemotherapy drugs. Five of these compounds were effective against blasts, but none were effective against LSCs (p-value: 0.0256), suggesting a possible mechanism for post-treatment relapse or primary refractoriness. LSCs were also resistant to mitomycin-C, an agent that induces DNA interstrand crosslinks and DNA breaks, in contrast to blasts that were variably sensitive. Of note, we identified 12 drugs from 8 classes defined by mechanism of action that may target LSCs, in some cases preferentially, when compared with blasts. Drugs effective in preferentially targeting LSCs included tyrosine kinase inhibitors, histone deacetylase inhibitors, 1 cyclin-dependent kinase inhibitor, 1 proteasome inhibitor and 1 microtubule assembly inhibitor. Several of the drugs that efficiently killed LSCs have been studied clinically in AML, while others have theoretical or established efficacy against LSCs by drug class. Only one commonly used drug in AML, sorafenib, a multikinase inhibitor used in FLT3+ disease that may improve survival in younger patients, was effective against LSCs. Blast specific drugs include romidepsin, dinaciclib, alvocidib, ganetespib, selinexor, dorsomorphin, vinblastine, cladribine, dabrafenib, selumetinib, etoposide, torkinib and those in Figure. Blast and LSC drug susceptibility patterns were distinct for each patient.Further, the engrafted xenograft subclone grew very rapidly, was resistant to standard chemotherapy, and possessed three new deleterious mutations in KMT2C (2), SF3B1 and 1 possibly damaging mutation in NUP214, suggesting possible genetic contributions to chemotherapy resistance. We also compared mutation profiles for LSCs vs. blasts in 5 patients, and identified LSC specific mutations in WNK3, WNK4 and BUB1, each in 2 of the 5, and there were also other mutations that were LSC or blast specific. Of note, Bub1 is a mitotic checkpoint serine/threonine kinase that controls mitosis in cancer stem cells (Venere et al Cancer Discov. 2013). WNK3 and WNK4 also both encode serine/threonine protein kinases.Conclusions:The distinct drug susceptibility patterns of patient-specific LSC and blast populations highlight the potential of an individualized approach to treat AML. LSCs are resistant to S-phase agents used in standard-of-care chemotherapy. Genetically distinct minority resistant LSC subclones present at diagnosis may grow rapidly under some conditions, and contribute to drug resistance and relapse. Incorporating the results of functional drug screening focused on LSC subclones may allow more individualized treatment of AML patients and identify patient-specific therapies that lead to improved outcomes. [Display omitted] DisclosuresCarson:Invivoscribe Inc.: Employment. Patay:Invivoscribe Inc.: Consultancy, Equity Ownership, Patents & Royalties. Becker:Novartis: Research Funding; Trovagene: Research Funding; CVS Caremark: Consultancy; JW Pharmaceuticals: Research Funding; Rocket Pharmaceuticals: Research Funding; Pfizer: Consultancy; Amgen: Research Funding; BMS: Research Funding; Abbvie: Research Funding; GlycoMimetics: Research Funding.

Ludwig Boltzmann Cluster Oncology (LBC ONC): first 10years and future perspectives.

SummaryIn 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.

Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission.

Chronic myeloid leukemia (CML) patients in sustained “deep molecular response” may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38− LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38− stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/μL, range 0.27–698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012–0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006–0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that “circulating” CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a “stem cell response” threshold to achieve and maintain TFR are ongoing.

Relationship of minimal residual disease (MRD) in acute lymphocytic leukemia (ALL) and putative leukemia stem cells (LSCs).

7012Background: MRD has emerged as a significant prognostic factor in ALL, but even levels < 0.1% still carry a risk of relapse. It is theorized that relapse is due to resistant LSCs, which survive...

Cell Density-Dependent Cytological Stage Profile and Its Application for a Screen of Cytostatic Agents Active Toward Leukemic Stem Cells.

Proliferation and expansion of leukemia is driven by leukemic stem cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in acute myeloid leukemia (AML) treatment. In this study, we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells, which include LSCs determining the overall stage profile. This change manifested in morphology, expression of both cell surface markers and redox-state proteins, as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Among the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of reactive oxygen species (ROS). Furthermore, it had low toxicity in vivo and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C-123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogeneic transplantation and/or ex vivo treatment preceding autologous transplantation.

Immature CML cells implement a BMP autocrine loop to escape TKI treatment

AbstractThe BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b+ cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance.

Abstract 91: Molecular insight into drug resistance in acute myeloid leukemia: An in-silico and in-vitro approach

Abstract INTRODUCTION: Drug resistance of leukemic stem cells is a surmountable obstacle to effective chemotherapy in acute myeloid leukemia (AML). P-glycoprotein (Pgp) and FLT3 undoubtedly contribute to worse prognosis and mechanisms of these proteins lead to shorter survival and chemotherapy resistance. Due to heterogeneity of stem cells in AML, outcome of patients with a normal karyotype is highly challenging and changes in drug resistance genes likely to be identified which will contribute to a better understanding of the disease biology, eventually leading to the development of alternative therapeutic approaches regarding drug resistance in AML. METHODOLOGY: Blood and Bone-marrow samples were collected from newly diagnosed adult AML patients with normal karyotype. Gene expression analysis of MDR genes like Pgp, MRP1, BCRP and LRP was done by qRT-PCR in FLT3-ITD+/- and CD34+/- subgroups. Higher expression of Pgp amongst all MDR genes instigated to undertake in-silico analysis of Pgp with natural compound library using YASARA. MTT assay was used to find out IC50 value of natural compounds shortlisted from in-silico analysis and used alone or with cytarabine on THP-1 cell line and in primary AML stem cells. Pgp inhibitory activity of natural compounds was assessed by fluorimetric MDR assay. RESULTS: A significant upregulation of Pgp expression was observed in FLT3-ITD+, CD34+ group after cytarabine exposure during their induction (7+3) therapy. Docking analysis revealed curcumin, hesperidin and silymarin having better binding affinity with Pgp as compared to its known inhibitor Verapamil. Furthermore, these selected compounds were used in-vitro, and results of MTT assay suggested 6.1µM, 40.73µM, 63.09µM and 54.95µM IC50 values of cytarabine, curcumin, hesperidin and silymarin respectively in THP-1 cell line. Combination index (CI) analysis revealed that curcumin (25µM), hesperidin (30µM) and silymarin (35µM) having synergistic effect with cytarabine and decreased IC50 value of cytarabine (1µM). In addition to this, Silymarin at 250nM and 500nM concentration showed equivalent inhibition of Pgp as compared to control Verapamil. CONCLUSION: A combination of in-silico and in-vitro screening revealed Curcumin, Hesperidin and Silibinin can be used as MDR modulators as well as chemosensitizer to reduce the cytotoxicity profile and drug resistance in AML. Citation Format: Urja Desai, Sheefa Mirza, Sonia Parikh, Rakesh Rawal. Molecular insight into drug resistance in acute myeloid leukemia: An in-silico and in-vitro approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 91. doi:10.1158/1538-7445.AM2017-91

CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide

Chemotherapy resistant leukemic stem cells (LSCs) are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP) of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952nM in HL60, 714nM in MOLT4] and relapsed patient blood samples with higher efficacy (85%) than IL2-TRAIL protein (46%). Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

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.

CX-01, a Low Anticoagulant Heparin, May Enhance Count Recovery and Treatment Efficacy in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) therapy is associated with pancytopenia and with a high failure rate due to resistant leukemia stem cells that home to marrow niches. We present here updated results of this study that combined the low anticoagulant heparin CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7+3) along with CX-01. Patients younger than 60 received consolidation therapy with high dose cytarabine along with CX-01. Twelve patients were enrolled (median age 56; range 22 - 74; 3 women). Three, 5, and 4 patients had good, intermediate, and poor risk disease, respectively. Two patients did not finish induction due to events unrelated to CX-01. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had a morphologic complete remission after one induction (CR1), including all patients with de novo AML (11/11). No patient required re-induction at Day 14. Eight patients are alive at a median follow-up of 24 months (range 13 - 29). Four patients remain in CR1. Four patients received an allogeneic stem cell transplant in CR1. All patients who received a full induction were evaluable for platelet recovery and had a median day to an untransfused platelet count ≥ 20,000/μl of 21. Five patients who received a full induction without myelopoietic growth factor support were evaluable for neutrophil recovery and had a median time to a neutrophil count ≥ 500/mL of 22 days (mean 22.6 ± 2.4, range 21 - 27). Median disease free survival was 14.8 months. Median overall survival was not attained at the maximum follow up time of 29.4 months. No CX-01-associated serious adverse events occurred. In a supplemental surface plasmon resonance study, CX-01 inhibited binding of CXCL12 to surface conjugated heparin with a 50% inhibitory concentration (IC50) of 4.7 nM, suggesting the possibility that CX-01 may enhance CR by disrupting the CXCL12-mediated leukemic stem cell marrow niche. We conclude that CX-01 is well tolerated when combined with intensive therapy for AML. It appears to be associated with enhanced count recovery and treatment efficacy. Thus, CX-01 warrants further study in this setting. DisclosuresKovacsovics:Seattle Genetics: Research Funding. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kennedy:Cantex Pharmaceuticals: Equity Ownership. Marcus:Cantex Pharmaceuticals: Employment, Equity Ownership. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Abstract Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated &amp;gt;50% inhibition, in another 40% it was 20-50% and in 20% inhibition was &amp;lt;20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p&amp;lt;0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML. Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3338.

Regain control of p53: Targeting leukemia stem cells by isoform-specific HDAC inhibition.

Leukemia stem cells (LSCs) are self-renewable, leukemia-initiating populations that are often resistant to traditional chemotherapy and tyrosine kinase inhibitors currently used for treatment of acute or chronic myeloid leukemia. The persistence and continued acquisition of mutations in resistant LSCs represent a major cause of refractory disease and/or relapse after remission. Understanding the mechanisms regulating LSC growth and survival is critical in devising effective therapies that will improve treatment response and outcome. Several recent studies indicate that the p53 tumor suppressor pathway is often inactivated in de novo myeloid leukemia through oncogenic-specific mechanisms, which converge on aberrant p53 protein deacetylation. Here, we summarize our current understanding of the various mechanisms underlying deregulation of histone deacetylases (HDACs), which could be exploited to restore p53 activity and enhance targeting of LSCs in molecularly defined patient subsets.

Identification of a New AHI-1-Mediated-PP2A-Beta-Catenin-BCR-ABL-JAK2 Complex Modulating Resistance of CML Stem/Progenitor Cells to Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of early phase CML patients. However, TKI monotherapies are not curative and initial and acquired resistance remain challenges. Particularly, leukemic stem cells (LSCs) are less responsive to TKIs and are a critical target population for TKI resistance. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is highly upregulated in LSCs and interacts with multiple kinases, including BCR-ABL and JAK2. We further showed that AHI-1-mediated complexes contribute to enhanced transforming activity of BCR-ABL and drug resistance of LSCs, suggesting AHI-1 as a new therapeutic target in CML. By screening the Prestwick Chemical Library, we have recently identified a specific growth inhibitory compound that potentially targets AHI-1: Cantharidin (CAN), an inhibitor of protein phosphatase 2A (PP2A). PP2A is a family of serine/threonine phosphatases that regulate cell signaling cascades involved in proliferation and cell cycle control of cancer cells. Evidence suggests the dysfunction of specific PP2A protein complexes is primarily responsible for the changes that lead to cell transformation. It was also reported that PP2A activity is downregulated in CML cells, particularly in blast crisis CML, due to overexpression of SET, and that the use of a PP2A activator inhibits the growth of CML cells. Interestingly, we have now demonstrated that CAN inhibits the growth of AHI-1-transduced BCR-ABL+ cells by about 30% compared to control cells, but this effect was significantly enhanced to 93% with the addition of imatinib (IM). As well, AHI-1-suppressed cells were more sensitive to CAN treatment. PP2A is highly expressed in K562 and IM-resistant K562 cells and combination treatment with CAN and TKI prevents growth and induces apoptosis in these cells more effectively than single treatments (2-3 fold, p<0.001). The combination also greatly reduced colony formation (CFC) of CD34+ CML cells, but CAN also inhibited CFCs of CD34+ normal bone marrow (BM) cells. To overcome toxicity issues, we have recently evaluated new, pre-clinically validated PP2A inhibitors, LB100 and LB102. These specifically suppress PP2A activity up to 75% in CML cells, as determined by immunoprecipitation (IP) phosphatase assays, and inhibit growth of these cells. Importantly, PP2A inhibitors plus TKIs significantly reduced the yield of colonies from CD34+ CML stem/progenitor cells compared to any single agent or combination of TKIs and this enhanced effect was most pronounced on cells from IM nonresponders (p<0.05). New PP2A inhibitors are also much less toxic to CD34+ normal BM cells compared to CAN (2-3 fold, p<0.001). Long-term culture-initiating cell assays showed that more primitive cells were eliminated to a greater extent by combination treatments. Moreover, the combination resulted in strong synergy in IM-insensitive cells (CI value <0.5). Notably, cell cycle analysis showed that treatment with LB100/LB102 alone induces a shift from G1 to G2/M phase (3-fold, p<0.05). A similar shift in the cell population was observed after combination treatment with IM, suggesting that the G2/M phase arrest is solely due to PP2A inhibition. Confocal microscopy showed the G2/M arrest, leading to mitotic catastrophe. Mechanistically, we have identified the PP2A subunit B (PR55a) as an AHI-1 interacting protein using IP/mass spectrometry; this was further confirmed in AHI-1-transduced cells by IP-Western analysis. Most interestingly, Western blot analysis showed that the combination treatment not only inhibits tyrosine phosphorylation of BCR-ABL, JAK2, STAT5, AKT and P38, but also significantly inhibits protein expression of these proteins compared to single agents. Inhibition of protein expression of AHI-1 and β-catenin by the combination was also observed and a new protein interaction between AHI-1 and β-catenin was identified. Moreover, the combination directly affects PP2A-mediated β-catenin dephosphorylation and BCR-ABL-mediated phosphorylation of β-catenin in CML cells, which leads to protein degradation of multiple proteins. In summary, we have uncovered new AHI-1-PP2A (PR55a) and AHI-1- β-catenin interactions and simultaneously targeting both BCR-ABL and PP2A activities in CML LSCs may offer an important new therapeutic possibility, through destabilization of the protein-protein interactions mediated by AHI-1. DisclosuresNo relevant conflicts of interest to declare.

Deletion of Pleiotrophin (PTN), a Vascular Niche Paracrine Factor, Abrogates Chronic Myeloid Leukemia Pathogenesis In Vivo

Tyrosine kinase inhibitor (TKI) therapy revolutionized the treatment of patients with chronic myeloid leukemia (CML), but TKI therapy alone is not curative. Leukemic stem cells (LSCs) are considered to be resistant to TKI therapy in CML, although the mechanisms which confer LSC resistance to TKI-mediated eradication remain poorly understood. Our laboratory has previously described the role of PTN, a paracrine factor which is secreted by bone marrow endothelial cells (BM ECs) and is necessary for the self-renewal and regeneration of normal hematopoietic stem cells (HSCs) in vivo. We hypothesized that PTN signaling in the BM vascular niche might also be important in CML pathogenesis and perpetuation of LSCs. In order to test our hypothesis, we crossed SCL-tTA/BCR-ABL mice, which develop CML over time under the control of doxycycline-treatment, with either PTN-/- or PTN+/+ mice, and compared CML pathogenesis in vivo. SCLtTA/BCR-ABL;PTN+/+ mice displayed evolution of chronic phase CML (splenomegaly, increased WBCs, left-shifted myelopoiesis, decreased survival) within 90-120 days. In contrast, SCLtTA/BCR-ABL;PTN-/- mice displayed normal range WBCs, decreased splenomegaly and significantly increased survival over the same time period. At 300 days post-induction of BCR-ABL, 33% percent of SCLtTA/BCR-ABL;PTN-/- mice remain alive compared to only 7% of SCLtTA/BCR-ABL;PTN+/+ mice (p<0.0001). Importantly, treatment of human CML cells with a specific anti-PTN antibody (BD Biosciences) significantly decreased CML colony formation in methylcellulose (P=0.02), whereas normal human CD34+ cells were unaffected. This result suggested the novel possibility that human CML cells may produce PTN in an autocrine manner as a means to propagate disease. These studies suggest that genetic or pharmacologic antagonism of PTN signaling is a potent strategy to inhibit CML pathogenesis. Ongoing studies will determine if PTN-targeted therapy, with or without TKIs, can mediate the eradication of LSCs which are responsible for perpetuation of this disease. DisclosuresHimburg:Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

O-desulfated heparin and chemotherapy for the treatment of AML.

7053 Background: Acute myeloid leukemia (AML) therapy is associated with pancytopenia and a high failure rate due to resistant leukemia stem cells that use CXCL12/CXCR4 to home to marrow niches. Pl...

Divergent Lineage-Specific Functions of PP2A in Chronic Phase CML and Lymphoid Blast Crisis

Background & Hypothesis: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase and functions as negative regulator of Akt- and Erk-signaling. PP2A attenuates downstream signaling of BCR-ABL1 and thereby functions as important tumor suppressor in chronic myeloid leukemia (CML). PP2A-activating drugs have been proposed for the treatment of CML and eradication of tyrosine kinase inhibitor resistant leukemia stem cells in CML (Neviani et al., 2013). Studying Cre-mediated ablation of PP2A in mouse models for BCR-ABL1-driven leukemia including myeloid CML in chronic phase (CML-CP), B lymphoid blast crisis (LBC) and B cell lineage Ph+ ALL, we were able to provide genetic evidence for this treatment concept in CML. Seemingly paradoxically, however, inducible ablation of PP2A in B cell lineage LBC and Ph+ ALL cells has the opposite effect and induced rapid cell death. These findings highlight fundamental differences in the regulation of Akt- and Erk-signaling in myeloid lineage and B cell lineage cells and have implications for specific therapy concepts that distinguish between chronic phase CML and lymphoid blast crisis/ Ph+ ALL.Results: Interestingly, PP2A protein levels are significantly higher in B cell lineage Ph+ ALL and CML lymphoid blast crisis (LBC) compared to chronic phase CML. To explore the function of PP2A during early B cell development, we deleted the α isoform of the PP2A subunit A in pro-B cells by introducing Mb1-Cre into Ppp2r1afl/fl mice. Analysis of bone marrow from Ppp2r1afl/flMb1-Cre (PP2A-cko) mice showed obvious B cell development block at the pre-B cell receptor checkpoint, although V(D) recombination and RAG1/2 activity were intact in PP2A-cko pre-B cells. Since high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL, we studied the function of PP2A in mouse models for B cell lineage ALL including Ph+ ALL and CML lymphoid blast crisis (LBC). After Cre-induced deletion of Ppp2r1a, we observed reduced protein expression of both PP2A subunit A and subunit C accompanied with decreased PP2A phosphatase. The PP2A-cKO ALL cells showed normal proliferation but significantly impaired colony formation capability and increased apoptosis, both of which could be rescued by overexpression of wildtype PP2A in PP2A-cKO ALL cells. We next transplanted luciferase expressing PP2A-KO ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although all of those mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles suggesting that these mice died from clones that had escaped Cre-mediated deletion of PP2A. Cre-mediated deletion increased phosphorylation levels of Stat5 and Erk, resulted in accumulation of p53 and increased output of the PI3K-AKT signaling pathway as measured by increased phosphorylation of FoxO factors, p70S6K and S6 ribosomal protein.Based on the involvement of PI3K-AKT in glucose metabolism, we measured the effects of inducible PP2A-deletion on glycolytic function in CML and Ph+ ALL/LBC cells. Interestingly, inducible deletion of PP2A induced profound imbalances of glucose metabolism in B cell lineage Ph+ ALL/LBC but not myeloid lineage CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-KO ALL cells indicated impaired balance of glycolytic flux and may account for increased apoptosis of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-cKO cells. In agreement with reported tumor suppressor role of PP2A in CML cells (Neviani et al., 2005), we observed that CEBPα-driven lineage conversion of B cell lineage Ph+ ALL/LBC into not myeloid lineage.Conclusion: Here we reported the requirement of PP2A in normal B cell development and lineage-specific oncogenic role of PP2A in BCR-ABL1-driven leukemia. Divergent roles of PP2A in Ph+ ALL/LBC and myeloid lineage CML may lead to future insights into mechanisms of LBC-transformation of CML and lineage-specific requirement of BCR-ABL1-transformation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 662: Generation and characterization of antibodies specific for IL1RAP antigen to target quiescent and proliferating AML leukemic stem cells

Abstract We have isolated and characterized blast cells and leukemic stem cells (LSCs) enriched from AML patients and identified cell surface targets selectively expressed on AML blast cells and LSCs and not their normal hematopoietic stem cell counterparts. A series of potent, specific, monoclonal antibodies (mAbs) have been generated against one of these AML LSC targets, IL1RAP. IL1RAP mAb clones were characterized for patient binding properties and also evaluated in in vitro cell based assays and in in vivo tumor xenograft models. These monoclonal antibodies demonstrated promising binding profiles in primary AML patients’ samples and robust anti-tumor activity in in vitro cell based assays and in vivo tumor xenograft models. Specifically, select clones have a high degree of specific binding to AML primary patient samples (&amp;gt;90%), and specifically kill primary patient AML cells in complement-dependent cytotoxicity assays. These IL1RAP clones are being evaluated for inhibition of clonogenic potential of AML patient samples in vitro in colony forming assays and against normal clonogenic hematopoietic bone marrow derived stem cells. Select IL1RAP mAb clones evaluated in orthotopic AML tumor xenograft models have shown potent inhibition of tumor engraftment as well as significant tumor growth inhibition effects ranging from 80-90%. Because a key driver of LSC resistance is their quiescence, we also evaluated the antitumor effect of IL1RAP mAbs on quiescent AML cells in vitro. In summary, the patient binding profiles and in vitro and in vivo efficacy against AML cells indicates that targeting IL1RAP is a promising therapeutic approach for the treatment of AML and has the potential for effective eradication of AML LSCs and long term remission in the clinic. Citation Format: Ping Jiang, Sharmili Roy, Jen Huang, Trang Dao-Pick, Jennifer Lu, Madhavi Mishra, Jeffrey Lin, Eric D. Hsi, Robert J. Tressler, Holger Karsunky. Generation and characterization of antibodies specific for IL1RAP antigen to target quiescent and proliferating AML leukemic stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2014-662

PTEN Regulates BCRP/ABCG2 and the Side Population through the PI3K/Akt Pathway in Chronic Myeloid Leukemia

A small population of cancer stem cells named the “side population” (SP) has been demonstrated to be responsible for the persistence of many solid tumors. However, the role of the SP in leukemic pathogenesis remains controversial. The resistance of leukemic stem cells to targeted therapies, such as tyrosine kinase inhibitors (TKIs), results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ATP-binding cassette sub-family G member 2 (ABCG2), is well known as a specific marker of the SP and could be controlled by several pathways, including the PI3K/Akt pathway. Our data demonstrated that compared with wild-type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated phosphatase and tensin homologue deleted on chromosome -10 (PTEN) and activated phosphorylated-Akt (p-Akt). PTEN and p-Akt downregulation could be abrogated by both the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin. Moreover, in CML patients in the accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression was accompanied by the loss of PTEN protein and the up-regulation of p-Akt expression. These results suggested that the expression of ABCG2 and the SP may be regulated by PTEN through the PI3K/Akt pathway, which would be a potentially effective strategy for targeting CML stem cells.

HLA Ligandome Analysis Of Chronic Myeloid Leukemia (CML), Revealed Novel Tumor Associated Antigens For Peptide Based Immunotherapy

Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Specific T-cell based immunotherapies for patients with CML might be able to eliminate these residual CML cells. For this goal the identification of tumor associated HLA-presented peptides, which are able to induce a tumor-specific T cell response, is indispensable. However, only few tumor associated antigens for CML are described till date. Thus the aim of this study was to identify for the first time naturally processed and presented HLA ligands from the cell surface of primary CML cells. HLA class I ligands from primary CML cells as well as from bone marrow mononuclear cell (BMNCs) and peripheral blood PBMCs of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. So far 10 CML patients (8 chronic phase, 2 accelerated phase) and 30 healthy donors were analyzed within this study. We were able to identify a total of more than 8200 CML derived HLA ligands representing >4500 different source proteins, of which 734 were exclusively represented in CML, but not in healthy PBMCs/BMNCs. 55 of these CML exclusive antigens are presented on 3 or more of all examined CML patients, representing, as broadly represented LiTAAs, promising targets for peptide vaccination. For the first time, previously predicted CML tumor associated antigens for example Myeloperoxidase (10 peptide sequences on 7 CML patients) and Proteinase 3 (5 peptide sequences on 4 CML patients) were here confirmed by direct elution from primary CML cells, which also served to validate our methodological approach. Notably, beyond that we also identified a vast array of novel proteins (e.g. Carcinoembryonic antigen-related cell adhesion molecule 8, CEACAM8; Matrix metallopeptidase 8, MMP8; intracellular adhesion molecule 3, ICAM3) that are broadly and exclusively represented in the ligandome of CML cells.By providing for the first time HLA class I tumor associated antigens, directly obtained from the HLA ligandomes of CML patients, this study may pave the way for the future development of a multipeptide-based immunotherapy approach to eradicate residual CML cells after conventional TKI therapy. Disclosures:No relevant conflicts of interest to declare.

PTEN Regulated BCRP/ABCG2 and Side Population Through PI3K/Akt Pathway In Chronic Myeloid Leukemia

A small population of cancer stem cells named as “side population (SP)” has been demonstrated to be responsible for many solid tumor maintenance. However, the role of SP in leukemic pathogenesis is still controversial. The resistance of leukemic stem cells in response to targeted therapies such as tyrosine kinase inhibitors (TKIs) results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ABCG2, is well-known as a specific marker of SP and could be controlled by several pathways including PI3K/Akt. Our data demonstrated that compared to wild type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated PTEN and activated p-Akt. It could be abrogated by both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin. Moreover, in CML patients at accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression accompanied with loss of PTEN protein and up-regulation of p-Akt expression was observed. These results suggested that expression of ABCG2 and fraction of SP may be regulated by PTEN through PI3K/Akt pathway, which will be the potentially effective strategy for targeting CML stem cells. Disclosures:No relevant conflicts of interest to declare.