Leukemic Stem Cell Population Research Articles

Venetoclax for AML: changing the treatment paradigm

AbstractVenetoclax is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore activation of apoptosis in malignancies, the survival of which depends on dysregulation of this pathway. Preclinical data, using various model systems including cell lines and patient samples, suggested targeting BCL-2 could be a successful therapeutic strategy in patients with acute myeloid leukemia (AML). As predicted by this work, the use of venetoclax in the clinical setting has resulted in promising outcomes for patients with this disease. Although venetoclax showed limited activity as a single agent in the relapsed disease setting, recent studies have shown that when combined with a backbone therapy of a hypomethylating agent or low-dose cytarabine, high response rates with encouraging remission durations for older patients with newly diagnosed AML who were not candidates for intensive induction chemotherapy were observed. Furthermore, venetoclax-based therapies allowed for rapid responses and were able to effectively target the leukemia stem cell population. Here we review the preclinical data that supported the development of venetoclax in AML, as well as the results of the promising clinical trials.

Human telomerase reverse transcriptase depletion potentiates the growth-inhibitory activity of imatinib in chronic myeloid leukemia stem cells.

Although tyrosine kinase inhibitors (TKIs) revolutionized the management of chronic myeloid leukemia (CML), resistance against TKIs and leukemia stem cell (LSC) persistence remain a clinical concern. Therefore, new therapeutic strategies combining conventional and novel therapies are urgently needed. Since telomerase is involved in oncogenesis and tumor progression but is silent in most human normal somatic cells, it may be an interesting target for CML therapy by selectively targeting cancer cells while minimizing effects on normal cells. Here, we report that hTERT expression is associated with CML disease progression. We also provide evidence that hTERT-deficient K-562cells do not display telomere shortening and that telomere length is maintained through the ALT pathway. Furthermore, we show that hTERT depletion exerts a growth-inhibitory effect in K-562cells and potentiates imatinib through alteration of cell cycle progression leading to a senescence-like phenotype. Finally, we demonstrate that hTERT depletion potentiates the imatinib-induced reduction of the ALDH+-LSC population. Altogether, our results suggest that the combination of telomerase and TKI should be considered as an attractive strategy to treat CML patients to eradicate cancer cells and prevent relapse by targeting LSCs.

STAT3 Plays a Critical Role in Mitochondrial Function and Survival of Primary AML Cells

Introduction: Acute myeloid leukemia (AML) is an aggressive disease with a dismal prognosis. This is largely due to high relapse rates, which stem from our inability to eliminate leukemia stem cells (LSCs) with conventional chemotherapy. As we have gained a deeper understanding of the biology of LSCs, new targets against these chemo-resistant cells have surfaced. One such example is treatment using venetoclax/azacititine, a therapy that has significantly improved the outcome for these patients. Notwithstanding, some patients show resistance to all treatments, and developing a larger repertoire of agents that target LSCs remains an unmet need in this disease. One key vulnerability of LSCs is their dependence on oxidative phosphorylation (OXPHOS). Although signal transducer and activator of transcription 3 (STAT3) has been classically studied as a transcription factor that regulates self-renewal and proliferation, it has also been shown to play an essential role in OXPHOS via regulation of the electron transport chain (ETC). Given that STAT3 is commonly overexpressed in AML, and LSCs are dependent on OXPHOS, we hypothesized that STAT3 may be an effective target for eradication of LSCs. Methods: We have developed a novel small molecule inhibitor of STAT3, SF25. This compound, as well as genetic knockdown of STAT3, was employed to test the functional role of STAT3 in primary AML specimens. Flow cytometry, colony-forming potential, and engraftment of primary samples in PDX mouse models were performed to assess therapeutic efficacy. RNA-seq, seahorse assays, and metabolomics experiments were also performed to determine molecular mechanisms linked to targeting STAT3. Results: Our data shows that inhibition of STAT3 in primary AML samples leads to decreased cell viability, colony-formation and engraftment potential in xenograft models, while not affecting normal hematopoietic stem cells. This effect appears to be a result of mitochondrial dysfunction in LSCs, as seen by a significant decrease in oxygen consumption rate of STAT3 depleted cells. The mitochondrial dysfunction and reduction in OXPHOS is mediated by the downregulation of several mitochondrial and nuclear encoded genes that are important for oxidative phosphorylation, including several electron transport chain complex genes. Inhibition of STAT3 also affects glutaminolysis, as noted by metabolomics analysis of leukemia stem cells treated with STAT3 inhibitor. We suspect this effect is mediated by down-regulation of Myc upon STAT3 inhibition, which blocks glutamine conversion to glutamate, and leads to further decrease in TCA cycle intermediates. Conclusions: Acute myeloid leukemia is an aggressive disease, largely due to the presence of a chemo-resistant population of leukemia stem cells. LSCs highly depend on proper mitochondrial function and OXPHOS, a process that is partly regulated by STAT3 via multiple mechanisms. We propose that inhibition of STAT3 is therefore an effective way of eliminating this population, making this a promising new target in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

KLF4 Controls Leukemic Stem Cell Self-Renewal in MLL-AF9-Induced Acute Myeloid Leukemia

Acute myeloid leukemia (AML) develops from sequential mutations which transform hematopoietic stem and progenitor cells (HSPCs) in the bone marrow into leukemic stem cells (LSCs) which drive the progression of frank leukemia. Especially poor outcomes in elderly patients coupled with frequent relapse have led to a dismal 28.3% 5-year survival, warranting the need for innovative therapeutic approaches. Successful targeted therapy will selectively eliminate LSCs, which possess distinct characteristics enabling self-renewal and chemotherapeutic resistance, while sparing normal HSPCs. We theorized that KLF4, a zinc finger transcription factor, maintains key self-renewal pathways in LSCs due to its known importance in preserving stemness in embryonic and cancer stem cells. KLF4 alters gene transcription through its activating and repressing domains as well as remodeling chromatin through various epigenetic mechanisms, and work from our lab has demonstrated that loss of KLF4 in leukemia driven by the BCR-ABL fusion oncogene results in depletion of LSCs (Park et. al in revision) while enhancing self-renewal of hematopoietic stem cells. To address this hypothesis, mice featuring floxed Klf4 gene (Klf4fl/fl) were crossed with transgenic Vav-iCre mice to produce mice with hematopoietic-specific deletion of Klf4 (Klf4Δ/Δ). The murine t(9;11)(p21;q23) translocation (MLL-AF9 or MA9) transduction model has previously been shown to reflect clinical disease attributes, and represents the MLL-rearranged human patient subset with particularly poor prognosis and relatively higher levels of KLF4. Lin−Sca-1+c-Kit+ (LSK) cells from Klf4fl/fl and Klf4Δ/Δ mice were transduced with retrovirus containing MA9 and GFP reporter and transplanted into lethally-irradiated wild-type (WT) mice to generate trackable Klf4fl/fl and Klf4Δ/ΔAMLs. Recipients of both MA9Klf4fl/fl and Klf4Δ/Δ cells developed a rapid expansion of leukemic cells with myeloid immunophenotype by flow cytometric analysis (CD11b+Gr-1+; 68-91%), characterized as AML with latency of approximately 44.5 days. To quantify the defect induced by loss of KLF4 in the leukemic stem cell population, we performed secondary transplant of multiple limiting-dilution cell doses of primary transformed leukemic bone marrow from moribund mice. Klf4Δ/Δ AML mice exhibited significantly improved survival in all dose-cohorts, in some cases presenting no detectable leukemic cells at completion of monitoring (225 days). Limiting dilution analysis using the ELDA online software tool demonstrated a 7-fold reduction from 1 in 513 in Klf4fl/fl to 1 in 3836 in Klf4Δ/Δ AML bone marrow cells capable of leukemic initiation function (p<0.001), a hallmark of LSCs. Using the ERCre-tamoxifen inducible deletion system, Klf4 deletion 15 days post-transplant of AML significantly improved survival of Klf4Δ/Δ mice compared to controls, demonstrating KLF4 promotes maintenance of disease. Plating of leukemic bone marrow from Klf4Δ/Δ mice in methylcellulose medium revealed a reduction in serial colony-forming ability, further supporting a defect in self-renewal. To further determine the mechanisms connected to this reduction in functional LSCs, we isolated leukemic granulocyte-macrophage progenitors (L-GMPs), a population previously reported to be highly enriched for functional LSCs and representing a comparable cellular subset in human clinical samples, from Klf4fl/fl and Klf4Δ/Δ AMLs and conducted RNA-Seq to identify potential transcriptional targets of KLF4 with therapeutic promise. Taken together, these data suggest a novel function of the stemness transcription factor KLF4 in the preservation of leukemic stem cells in AML. Whereas prior models based on KLF4 expression in human cell lines and bulk AML samples have proposed a tumor suppressive role, our work suggests KLF4 supports expansion of leukemic cells with a stem cell phenotype and serial assays suggest an effect on LSC self-renewal. Further studies are being conducted to define the transcriptional and epigenetic mechanisms governing these findings. Understanding the molecular changes induced by loss of KLF4 presents promise for development of new therapies selectively targeting LSCs. Disclosures No relevant conflicts of interest to declare.

Characterization of Leukemic Stem Cells Heterogeneity in Chronic Myeloid Leukemia

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a subpopulation is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients using a selected panel of 96 primers. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). CITE-seq allows for unbiased, further in-depth transcriptome analysis as wells as immunophenotypic characterization by pre-staining cells with a panel of DNA-barcoded antibodies prior to sequencing. DNA-barcoded antibodies convert the protein expression into readable sequences through unique oligo-conjugates as identifiers. Using CITE-seq with a panel of 44 distinct surface markers designed to immunophenotypically differentiate between stem/progenitors cells and leukemic clones we simultaneously characterize the molecular and immunophenotypic heterogeneity within Lin-CD34+/Lin-CD34+CD38- CML stem/progenitor compartment at diagnosis. Additionally by comparing the LSCs transcriptome from patients with different therapeutic outcome after 12 months of therapy we describe how differences in heterogeneity and the presence of immunophenotypic therapy-insensitive LSCs at diagnosis (Lin-CD34+CD38-CD45RA-cKIT-CD26+) contribute to therapy response. Disclosures Richter: Novartis: Consultancy; Pfizer: Consultancy, Research Funding.

The Splicing Factor RBM17 Supports Leukemia Stem Cell Self-Renewal

Background: Acute myeloid leukemia (AML) is thought to be sustained by sub-populations of leukemia stem cells (LSCs), which possess the capacity for self-renewal and differentiation and are believed to be responsible for disease initiation, relapse and chemoresistance. There is therefore an urgent need to develop therapies that target the LSC population to achieve effective AML treatment. To date there has been limited success in this endeavor, highlighting the importance of gaining a more comprehensive understanding of the mechanistic elements that underpin LSC function. Aberrant alternative splicing is recognized as a key driver of cancer. In the context of AML, genome-wide sequencing studies have shown that approximately one third of genes are differentially spliced in primitive CD34+ cells in AML patients compared to those obtained from normal controls. In particular, LSCs also have a unique splicing profile when compared to normal aging HSCs, underscoring the importance of understanding the mechanistic controls of aberrant splicing in LSC function. Results: Previous studies examining the link between aberrant splicing and AML focused on spliceosome genes with somatic mutations in AML patients. To examine mechanisms that mediate aberrant alternative splicing in LSC beyond splicing factor mutations, we performed a data-mining survey of 203 known mRNA splicing factors. Strikingly, RNA-binding motif protein 17 (RBM17) is the only splicing factor that is both strongly linked to poor AML prognosis and significantly elevated in LSC-enriched subsets of primary AML samples based on a recent study of 78 AML samples with normal karyotype. RBM17 has been implicated in regulating alternative splicing and cancer chemotherapy resistance. However, its function in AML or LSCs is not known. From our studies, we found that the level of RBM17 protein is elevated in the phenotypically primitive subsets of primary AML samples (n=8, RBM17+%: 67.42% in CD34+ versus 34.66% in CD34-fractions). Depletion of RBM17 with shRNAs in 3 human primary AML samples resulted in reduced colony formation compared to shScramble controls. More importantly, knockdown of RBM17 in a primary AML sample greatly impeded AML engraftment in immune-deficient mice (mean of 0.89 control versus mean of 0.1671 and 0.3171 shRNAs), suggesting that RBM17 is required for the stem and progenitor potential of AML and maintenance of LSC populations. Intriguingly, in contrast to the situation for the malignant hierarchy, the level of RBM17 in normal HSCs is lower than that in more committed cell populations in the normal hematopoietic system. To determine if RBM17 plays different roles in malignant LSC and normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBM17 with shRNAs in human cord blood (CB) derived CD34+ HSPCs, and found RBM17 knockdown had negligible adverse impact on both CB total colony and primitive GEMM colony outputs and yielded no increase in apoptosis, indicating no defects were apparent to primitive cells of CB as read out in vitro. To uncover the molecular mechanisms underlying the role of RBM17 in LSC functions, we preformed RBM17 eCLIP-seq in the K562 and HL60 human leukemic cell lines. We then cross-analyzed the CLIP-seq datasets with a published ENCODE RNA-seq dataset (RBM17 knockdown in K562 cells), where we found RBM17 directly binds to transcripts of stem cell program-related genes and regulates the splicing of these genes, including MADD (MAP kinase activating death domain) and MRPS18C (mitochondrial ribosomal protein S18C). We further demonstrated that the splicing patterns of MADD and MRPS18C are mediated by RBM17 in primary AML samples. In our ongoing functional validation experiments, isoform-specific knockdown of MADD or MRPS18C splice variants downstream of RBM17 impeded colony forming capacity and induced myeloid differentiation in the MOLM13 AML cell line. These results suggest that RBM17-mediated splicing events impact primitive cell function in AML. Conclusion: We have identified RBM17 as a novel LSC-regulating factor, plays an important role in maintaining AML LSC function through regulating the alternative splicing of stem cell program-related genes. The potential LSC-selective role for RBM17, along with its downstream splicing events, represent promising putative therapeutic targets whose modulation could offer attractive therapeutic windows in AML treatment. Disclosures No relevant conflicts of interest to declare.

Targeted Killing of AML Cells By Bispecific Antibody Armed Activated T Cells

Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of hematopoietic stem/progenitor cells that poses a significant therapeutic challenge. Recurrence of the disease is attributed to leukemia-initiating cells, also referred to as leukemic stem cells (LSCs), which are thought to be spared from chemotherapy and capable of reinitiating the disease. Thus, novel non-toxic therapeutic strategies to target and eradicate LSCs and AML blasts are urgently needed. Immunotherapy (IT) is an attractive strategy to improve outcomes for patients with AML, as it does not rely on the cytotoxic mechanisms employed by conventional chemotherapies. Methods: Bispecific antibody was produced by chemical hetero-conjugation of anti-CD33 monoclonal antibody (gemtuzumab ozogamicin [GO]) linked to calicheamicin to anti-CD3 monoclonal antibody (anti-CD3 x GO bispecific antibody, CD33Bi) or anti-CD3 and anti-CD123 (CD123Bi). We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD33Bi and CD123Bi armed ATC using flow cytometry-based cytotoxicity assays. Cytokines and chemokines released during CD33Bi or CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. A xenogeneic NOD/SCID/gamma chain KO (NSG) mouse model was used for the evaluation of in vivo activity of Bispecific antibody (50ng/million cells) Armed activated T cells (BATs). Engraftment of AML cells-KG1 was tracked by quantifying the human CD45+ AML cells using flow cytometry. Two patient samples were analyzed for blast and LSC population before and after incubation with CD123-BATs. Results: In a non-radioactive quantitative flow cytometry-based cytotoxicity assay Bispecific antibody (50ng/million cells) Armed activated T cells (BATs) show substantial killing by both CD33-BATs and CD123-BATs against AML cell lines (EOL1, KG1, NoMo1 and TF-1) at 1:1 and 2:1 E/T ratios ranging from 60-90% cytotoxicity and produced increased levels of Th1 cytokines (TNF-alpha, GM-CSF and IFN-gamma). The KG1 engrafted 6- to 8-week-old NSG mice (n=8 mice/group) injected IV with 20 x 106 CD33-BATs (3x/week for 4 weeks; 100ml/injection) was able to inhibit leukemic cell expansion significantly compared to 0.06 mg/kg gemtuzumab ozogamicin [GO] antibody (p<0.0006); likewise significant inhibition of leukemic cell expansion was observed with CD123-BATs compared to PBS control mice (p<0.009). One patient sample incubated with CD123-BATs showed significant reduction in blast after CD123-BATs treatment compared to before treatment (13.2% vs. 30%) or CD34+CD38- LSC after CD123-BATs treatment (5.2%) compared to before treatment (20%). Second patient sample also showed 50% reduction in blast population, no LSC were detected in this sample. Conclusions: ATC armed with CD33Bi or CD123Bi at 50 ng/106 cells showed comparable cytotoxicity directed at either high or low leukemic blast populations, able to inhibit leukemic cell expansion significantly compared to the control mice. This approach may provide a potent and non-toxic strategy to target leukemic blasts and leukemic stem cells. Disclosures Huang: TransTarget Inc.: Other: Co-Founder.

A Phase 2 Study of Glasdegib for the Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients with High Risk of Post-Allogeneic Stem Cell Transplantation Relapse

A Phase 2 Study of Glasdegib for the Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients with High Risk of Post-Allogeneic Stem Cell Transplantation Relapse

Novel Therapeutics That Can Target and Eradicate Leukemic Stem Cells in Acute Myeloid Leukemia: Investigating FDA-Approved Anti-Inflammatory Compounds

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. The 5-year survival rate is 28% in patients aged 20 years and older and 64% in patients aged 19 years and younger (SEER 2019). A large portion of these patients succumb to the disease partially due to the chemo-resistant nature of leukemic stem cells (LSCs). Hence, novel therapies targeting unique LSC biology that spare hematopoietic stem cells (HSCs) are needed to eliminate and avoid reoccurrence of this disease. We had previously identified FDA-approved anti-inflammatory glucocorticoids mometasone, halcinonide, and budesonide as compounds that induce terminal differentiation of the LSC (CD34+CD38-) and progenitor cell (CD34+CD38+) populations to leukemic blast cells (CD15+CD34-) in refractory human AML (Laverdière & Boileau et al., Blood Can. J. 2018). Following the paradigm of successful differentiation treatment in AML (acute promyelocytic leukemia with all-trans retinoic acid), the effects and mechanism of action of the glucocorticoids on LSCs need to be further investigated for other AML subtypes. Furthermore, dexamethasone, a glucocorticoid currently used to successfully treat acute lymphoblastic leukemia (ALL), is being studied in a Phase II clinical trial for induction and post-remission chemotherapy in older patients with de novo or therapy-related AML (clinicalTrials.gov, NCT03609060). To identify the subtypes of AML that are sensitive to steroid-induced LSC differentiation, we began by screening a panel of cell lines (F36P, MOLM-13, Kasumi-6, Kasumi-1 and K562) and observed that only Kasumi-1, a pediatric leukemia carrying the t(8;21) mutation leading to the fused RUNX1-RUNX1T1 gene, was responsive to glucocorticoid treatment, although without differentiation. This is consistent with the finding of Simon et al. who observed a loss of bulk AML cells in RUNX1 AML samples following dexamethasone treatment (Simon et al., Clin Cancer Res. 2017). However, we observed expansion of bulk cells following differentiation of LSCs in primary AML, indicating different mechanisms of steroid response in different samples: differentiation of LSCs or overall loss of AML cells. We will further investigate these compounds in a panel of 10 genetically defined primary AML samples to classify which oncogenetic drivers or subtypes of AML are linked to sensitivity to the three glucocorticoids, including which drive cell death vs LSC differentiation. We will perform ex vivo and in vivo studies of the glucocorticoids to assess the extent of engraftment in treated versus DMSO treated samples. This additional data will be presented at the annual meeting. In addition, to explore the mechanism of action of these steroids in AML, we investigated the roles of the cytokines interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF), granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L), used to culture AML, on the differentiation effects induced by the glucocorticoids. We observed that only FLT3L was required for the complete differentiation of LSCs. In summary, we have observed that the three glucocorticoid steroids (mometasone, halcinonide, and budesonide), as well as dexamethasone to a lesser extent, can induce two different responses in a sample-dependent manner: terminal differentiation of LSCs or overall cell loss. We have also observed that the differentiation response requires FLT3L for maturation of the AML cells. Our current studies involve in vivo and genomic assays to determine the effect on functional LSCs and the genetic markers of sensitivity and we will present these results. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement.

Targeting CD70 with Cusatuzumab Eliminates Acute Myeloid Leukemia Stem Cells in Humans

Introduction Acute myeloid leukemia (AML) is a heterogenous hematological malignancy driven by leukemia stem cells (LSCs) (Lapidot et al, 1994). LSCs resistant against conventional chemotherapy represent the major cause of relapse. Elderly or unfit AML patients not eligible for intensive chemotherapy are treated in a palliative setting with hypomethylating agents (HMA) or low dose Ara-C, but responses are modest and not durable. The reason for the low efficacy of HMA treatment is their insufficient action on the disease initiating- and -maintaining LSC population (Craddock et al, 2013). We recently demonstrated that CD34+ AML cells (progenitors and LSC) consistently express the tumor necrosis factor family ligand CD70 as well as its receptor CD27 and that cell-autonomous CD70/CD27-signaling propagates the disease (Riether et al. 2017). The aim of the current study was to evaluate whether resistance to HMA treatment can be overcome by combining HMA with an anti-CD70 monoclonal antibody treatment. Experimental design The effect of HMA treatment on the expression of CD70 on primary human CD34+CD38- AML LSCs was determined in vitro cultures and in patients treated with HMA in vivo. The therapeutic potential of targeting CD70-expressing LSCs in presence and absence of HMA was assessed using the anti-CD70 ADCC-optimized monoclonal antibody (mAb), cusatuzumab, and an effector-dead anti-CD70 mAb in colony formation and re-plating assays as well as patient-derived xenograft models (Silence et al, 2014). The clinical relevance of the findings was determined in a clinical phase 1 trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine (AZA, NCT03030612). Four different dose levels of cusatuzumab (1, 3, 10 and 20 mg/kg Q2W) were studied; AZA was administered at 75 mg/m² for 7 days every 28 days. Results We found that resistance of AML LSCs to HMA treatment is mediated by the up-regulation of the CD70. The up-regulation of CD70 triggered cell-autonomous CD70/CD27 signaling on AML LSCs. Based on these findings we hypothesized that the upregulation of CD70 by HMA may render LSCs more susceptible to CD70-targeting interventions. Targeting CD70-expressing LSCs by a blocking anti-CD70 mAb and the anti-CD70 mAb cusatuzumab, which blocks CD70/CD27-signaling and additionally mediates ADCC and CDC, eradicated LSCs in colony and re-plating assays in vitro and in xenotransplantation experiments in vivo. HMA in combination with blocking αCD70 mAb synergistically reduced LSC numbers in vivo and this was even more efficient when ADCC-enhanced αCD70 mAb cusatuzumab was added in the presence of NK cells. In order to test the hypothesis that targeting CD70 in combination with HMA eliminates LSCs in AML patients, we initiated a phase 1 dose-escalation trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with azacitidine. No dose-limiting toxicities (DLT) were observed in the dose-escalation phase 1 trial and responses were observed across the dose levels (1-20 mg/kg). A single dose of cusatuzumab reduced bone marrow blasts in just two weeks in all patients on average by 32%. Cusatuzumab monotherapy significantly reduced LSC numbers and frequencies in all patients analyzed in the bone marrow as assessed in limiting dilution colony assays. Single cell sequencing analysis revealed that cusatuzumab induced gene signatures related to myeloid differentiation and apoptosis in LSCs. In combination with azacitidine, cusatuzumab induced CR/CRi in 10 out of 12 patients. Responses were observed at all dose levels of cusatuzumab and median time to response was 3.3 months. Conclusions Blocking CD70/CD27-signaling and targeting CD70-expressing LSCs by the ADCC-optimized mAb, cusatuzumab, eliminated LSCs in vitro and in xenotransplantation experiments. In a phase 1 study promising activity of cusatuzumab in combination with HMA was observed in AML patients, in which translational data indicate that cusatuzumab selectively eliminates CD70-expressing LSCs. Disclosures Van Rompaey: argenx: Employment, Equity Ownership, Patents & Royalties. Moshir:Argenx: Employment, Equity Ownership. Delahaye:argenx: Employment, Equity Ownership. Gandini:Argenx: Employment, Equity Ownership. Erzeel:argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Fung:argenx: Consultancy, Equity Ownership. De Haard:Argenx: Employment, Equity Ownership, Patents & Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents & Royalties.

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia.

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post‐treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients. © 2019 The Authors. Basic Protocol 1: Immunophenotypic LAIP detection for measurable residual disease monitoring Basic Protocol 2: Immunophenotypic detection of CD34+CD38− leukemic stem cells

Insulin-like growth factor (IGF) signaling in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer, characterized by an uncontrolled expansion and accumulation of T-cell progenitors. During leukemic progression, immature T cells grow abnormally and occupy the bone marrow compartment, thereby interfering with the production of normal blood cells. Pediatric T-ALL is curable with intensive chemotherapy, but there are significant, long-term side effects and ~20% of patients suffer relapse for which there are limited treatment options. Adult T-ALL in contrast is largely incurable and refractory/relapsed disease is common despite multi-agent chemotherapy (5-year overall survival of ~40%), and thus new therapeutic targets are needed. We have reported previously on the role of insulin-like growth factor (IGF) signaling in T-ALL, and shown that it exerts potent phenotypes in both leukemia stem cell and bulk tumor cell populations. Modulators of IGF signaling may thus prove useful in improving outcomes in patients with T-ALL. In this review, we summarize the most recent findings relating to IGF signaling in T-ALL and outline therapeutic options using clinically relevant IGF signaling modulators.

Leukemic stem cells hiding in plain sight

Activation of NK-mediated immune surveillance clears leukemic stem cell populations.

CHARACTERIZATION OF THERAPY-INSENSITIVE LSC SUBPOPULATIONS IN CHRONIC MYELOID LEUKEMIA

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a sub population is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent LSC-subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). By comparing LSCs from patients with different therapeutic outcome we describe how LSC heterogeneity at diagnosis correlate with long-term therapy response. We furthermore evaluate the potential for humanized ossicle-niche xenotransplantation (Reinisch et al., 2016) as a model system for functional characterization of LSC subpopulations in vivo.

Abstract CT103: Low-dose daunorubicin to target leukemia stem cells in newly diagnosed and relapsed/refractory AML

Abstract Background: Chemotherapy-resistant leukemia stem cells (LSC) are responsible for disease relapse in many patients with Acute Myeloid Leukemia (AML). The Wnt/β-catenin and PI3K/Akt pathways are frequently activated in cancer and interact to stimulate stem cell expansion through phosphorylation of β-catenin by Akt (pS552-β-catenin). We hypothesize that targeting pS552-β-catenin may inhibit chemoresistant LSCs. High-throughput screening identified that anthracyclines doxorubicin (DXR) and daunorubicin (DNR) inhibit Akt:β-catenin interaction at significantly lower doses than conventionally used. Using a mouse leukemia model, we repurposed DXR as a targeted therapy for pS552-β-catenin inhibition rather than a broadly cytotoxic agent. In contrast to the typical dose, which may stimulate expansion of resistant LSC, targeted/low-dose DXR inhibited chemoresistant LSC expansion and significantly reduced LSC tumorigenic potential. AML patients typically receive high-dose DNR but often relapse due to persistence of therapy-resistant LSCs, which frequently express pS552-β-catenin. Here, we use a metronomic approach to test the ability of low-dose anthracycline therapy to target therapy-resistant LSCs. Methods: A proof of concept trial (NCT02914977) was designed to measure the ability of low dose DNR to inhibit pS552-β-catenin in LSCs of adult relapsed/refractory AML patients. A protocol amendment added a second cohort of patients with newly diagnosed AML undergoing standard chemotherapy with DNR and cytarabine (7+3 induction). Treatment in each cohort consisted of DNR given at a dose of 6.75mg/m2/day x 5 days. Patients with relapsed/refractory AML had bone marrow aspiration pre-treatment prior to day 1, followed by DNR on days 1-5, and a post-treatment bone marrow aspiration on day 8. Patients with newly diagnosed AML were treated with conventional 7+3 chemotherapy. A sample from the day 14 bone marrow was collected. DNR was given on days 15-19. Bone marrow aspiration was performed with blood count recovery and samples sent for LSC marker assessment. In this study, we will determine the safety, pharmacokinetics, and pharmacodynamic effects of low dose DNR. The LSC population and phosphorylation status of β-catenin will be measured pre- and post-treatment. Two or more prior induction attempts are required for study entry for refractory patients; relapsed patients require only one prior induction. The trial has enrolled 14 patients: 11 in the relapsed cohort and 3 in the newly diagnosed cohort. A total of 18 patients are planned. In this pilot proof of concept trial, we will demonstrate the feasibility of serial bone marrow aspiration to measure biomarkers of LSC response. This clinical proof of concept trial design with serial bone marrow aspirations may also be used to rapidly assess the validity of other LSC markers and targeted agents. Citation Format: Tara L. Lin, John M. Perry, Xi He, Gregory Reed, Na Zhang, Scott Weir, Joseph McGuirk, Linheng Li. Low-dose daunorubicin to target leukemia stem cells in newly diagnosed and relapsed/refractory AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT103.

Energy metabolism and drug response in myeloid leukaemic stem cells.

Despite significant advances in the treatment of myeloid malignancies, many patients become resistant to therapy and ultimately succumb to their disease. Accumulating evidence over the past several years has suggested that the inadequacy of many leukaemia therapies results from their failure to target the leukaemic stem cell (LSC). For this reason, the LSC population currently represents the most critical target in the treatment of myeloid malignancies. However, while LSCs are ideal targets in the treatment of these diseases, they are also the most difficult population to target. This is due to both their heterogeneity within the LSC population, and also their phenotypic similarities with normal haematopoietic stem cells. This review will highlight the current landscape surrounding LSC biology in myeloid malignancies, with a focus on altered energy metabolism, and how that knowledge is being translated into clinical advances for the treatment of chronic and acute myeloid leukaemia and myelodysplastic syndromes.

PS1339 UNSUPERVISED HIERARCHICAL CLUSTERING OF PRE‐TREATMENT IMMUNOPHENOTYPIC FEATURES IDENTIFIES MDS PATIENTS AT HIGH RISK OF DISEASE PROGRESSION AFTER ALLOGENEIC TRANSPLANTATION

Background:Myelodysplastic syndrome (MDS) and myelodysplatic/myeloproliferative neoplasm (MDS/MPN) belong to a heterogeneous group of bone marrow (BM) diseases. Immunophenotyping is part of the basic diagnostic workup, but the biological implications and clinical consequences of distinct surface antigen expression patterns in MDS have not been reported.Aims:To assess pre‐treatment immunophenotypic features in MDS patients (pts) and to identify associated disease characteristics and outcome.Methods:We analyzed 175 pts with MDS (n = 155) or MDS/MPN (n = 20) who received an allogeneic cell transplantation (HCT, median age 59.2 [range 18.5–74.4] years) using peripheral blood (n = 174) or cord blood (n = 1) stem cells after myeloablative (n = 45), reduced‐intensity (n = 58) or non‐myeloablative (n = 72) conditioning. Donors were matched related (n = 31) or unrelated (n = 96) or had at least one allelic mismatch (n = 48). Prior to HCT, 53% of pts received cytoreductive therapy (24% azacitidine, 21% intensive chemotherapy, 8% both). Pts were grouped according to IPSS‐R (0% very good, 8% good, 31% intermediate, 25% poor, 37% very poor). Flow cytometric analysis was performed centrally on pre‐treatment bone marrow mononuclear cells. Median follow up after HCT was 3.9 years.Results:We identified four pts subgroups with distinct pre‐treatment immunophenotypic features by unsupervised hierarchical clustering (Figure 1A). For pts in the first cluster (n = 12) a particularly adverse outcome after HCT was observed. Compared to all other clusters, this cluster was characterized by a higher expression of immature surface antigens (CD34, P < .001; CD117, P < .001) and a higher expression of the leukemic stem cell population CD34+/CD38‐ (P = .001), of myeloid antigens (CD11b, P = .05; CD13 P < .01; CD33, P < .01; CD15, P < .01; CD65, P = .05 and CD64, P < .01), of pan‐leukocyte antigens (CD45, P = .03) and a lower expression of T‐cell antigens (CD2, P < .01 & CD7, P < .01). Pts in cluster 1 were more likely to present with an excess of blasts (P = .03) and to harbor a complex karyotype (KT, P = .02). Although pts in cluster 1 also more often received cytoreductive treatment prior to HCT (P = .04) they had a higher cumulative incidence of relapse/progression (CIR, P < .01, Figure 1B) including a higher incidence of secondary acute myeloid leukemia (CIsAML, P < .01, Figure 1C). This adverse prognostic impact was also seen in multivariate analyses for CIR after adjustment for age at HCT and IPSS‐R (Hazard ratio [HR] 3.2, 95% Confidence interval [CI] 1.1–9.6, P = .04) and for CIsAML after adjustment for excess of blasts and blast count prior to HCT (HR 3.2, 95% CI 1.0–9.6, P = .04). Cluster 1 immunophenotypic features also remained predictive for higher CIR and CIsAML in bivariate analyses including the presence of a monosomal (HR 2.9, P = .04 & HR 3.4, P = .02, respectively) or complex KT (HR 2.6, P = .08 & HR 3.0, P = .04, respectively).Summary/Conclusion:Assessment of pre‐treatment surface antigen expression patterns allows for identifying a subgroup of MDS pts with adverse clinical outcome after HCT irrespective of clinical prognosticators or sensitivity to pre‐HCT treatment approaches. This subgroup of pts requires post‐HCT strategies to maintain disease control.image

Assessing the link between epigenetics and metabolism in acute myeloid leukemia

Abstract Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to generate the many myeloid and lymphoid cells. When dysregulated, cancers such as Acute myeloid leukemia (AML) may develop. AML is characterized by expansion and proliferation of undifferentiated myeloid cells that derive from a Leukemic Stem Cell (LSC) population. AML is the most common form of leukemia with 19,520 new cases in 2018, with an adult five-year survival rate of 24%. Here, we examine a newfound relationship between glutamine metabolism, epigenetics, and targeted differentiation of LSCs in AML to promote disease clearance. Glutamine is catalyzed by glutaminase (GLS) to supply α-ketoglutarate (αKG) to the tricarboxylic acid (TCA) cycle and as substrate to demethylase enzymes. We hypothesize that glutamine metabolism plays an integral role in balancing the fate decision of LSCs and that targeting GLS will impact epigenetic modifications and gene expression to affect cell fate. Glutaminase knockout may also be exploited to promote differentiation of AML LSCs. Preliminary data demonstrates the link between epigenetics and metabolism in AML. We profiled three different AML cell lines’ metabolic responses to INCB54329, an inhibitor of bromodomain proteins. We present a heterogeneous response to INCB54329 in oxygen consumption rate, extracellular acidification rate, reactive oxygen species generation, and mitochondrial mass and membrane potential. The precise function of glutaminolysis in hematopoiesis remains unclear. Unraveling the link between glutamine metabolism, epigenetic modifications, and stem cell function presents a novel therapeutic opportunity for various hematologic malignancies, chief among them acute myeloid leukemia.

BETP degradation simultaneously targets acute myelogenous leukemia stem cells and the microenvironment.

Anti-leukemic effect of BET/BRD4 (BETP) protein inhibition has been largely attributed to transcriptional downregulation of cellular anabolic/anti-apoptotic processes but its effect on bone marrow microenvironment, a sanctuary favoring persistence of leukemia stem/progenitor cells, is unexplored. Sustained degradation of BETP with small-molecule BET proteolysis-targeting chimera (PROTAC), ARV-825, resulted in marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interaction, in AML cells. Abrogation of surface CXCR4 expression impaired SDF-1α directed migration and was mediated through transcriptional down-regulation of PIM1 kinase that in turn phosphorylates CXCR4 and facilitates its surface localization. Down-regulation of CD44/CD44v8-10 impaired cystine uptake, lowered intracellular reduced glutathione and increased oxidative stress. More importantly, BETP degradation markedly decreased CD34+CD38-CD90-CD45RA+ leukemic stem cell population and alone or in combination with Cytarabine, prolonged survival in mouse model of human leukemia including AML-PDX. Gene expression profiling and single cell proteomics confirmed down regulation of the gene signatures associated with 'stemness' in AML and Wnt/β-catenin, Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell intrinsic signaling, stromal interactions and metabolism in AML.

Mesenchymal Niche-Specific Expression of Cxcl12 Controls Quiescence of Treatment-Resistant Leukemia Stem Cells.

Chronic myeloid leukemia (CML) originates in a hematopoietic stem cell (HSC) transformed by the breakpoint cluster region (BCR)-abelson (ABL) oncogene and is effectively treated with tyrosine kinase inhibitors (TKIs). TKIs do not eliminate disease-propagating leukemic stem cells (LSCs), suggesting a deeper understanding of niche-dependent regulation of CML LSCs is required to eradicate disease. Cxcl12 is expressed in bone marrow niches and controls HSC maintenance, and here, we show that targeted deletion of Cxcl12 from mesenchymal stromal cells (MSCs) reduces normal HSC numbers but promotes LSC expansion by increasing self-renewing cell divisions, possibly through enhanced Ezh2 activity. In contrast, endothelial cell-specific Cxcl12 deletion decreases LSC proliferation, suggesting niche-specific effects. During CML development, abnormal clusters of colocalized MSCs and LSCs form but disappear upon Cxcl12 deletion. Moreover, MSC-specific deletion of Cxcl12 increases LSC elimination by TKI treatment. These findings highlight acritical role of niche-specific effects of Cxcl12 expression in maintaining quiescence of TKI-resistant LSC populations.