Leukemia-initiating Cell Activity Research Articles

The circadian clock circuitry modulates leukemia initiating cell activity in T-cell acute lymphoblastic leukemia

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, characterized by restricted cellular subsets with asymmetrically enriched leukemia initiating cell (LIC) activity. Nonetheless, it is still unclear which signaling programs promote LIC maintenance and progression.MethodsHere, we evaluated the role of the biological clock in the regulation of the molecular mechanisms and signaling pathways impacting the cellular dynamics in T-ALL through an integrated experimental approach including gene expression profiling of shRNA-modified T-ALL cell lines and Chromatin Immunoprecipitation Sequencing (ChIP-Seq) of leukemic cells. Patient-derived xenograft (PDXs) cell subsets were also genetically manipulated in order to assess the LIC activity modulated by the loss of biological clock in human T-ALL.ResultsWe report that the disruption of the circadian clock circuitry obtained through shRNA-mediated knockdown of CLOCK and BMAL1 genes negatively impacted the growth in vitro as well as the activity in vivo of LIC derived from PDXs after transplantation into immunodeficient recipient mice. Additionally, gene expression data integrated with ChIP-Seq profiles of leukemic cells revealed that the circadian clock directly promotes the expression of genes, such as IL20RB, crucially involved in JAK/STAT signaling, making the T-ALL cells more responsive to Interleukin 20 (IL20).ConclusionTaken together, our data support the concept that the biological clock drives the expression of IL20R prompting JAK/STAT signaling and promoting LIC activity in T-ALL and suggest that the selective targeting of circadian components could be therapeutically relevant for the treatment of T-ALL patients.

A fetal tumor suppressor axis abrogates MLL-fusion-driven acute myeloid leukemia.

MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r can be sufficient to drive transformation. However, despite the prenatal origin of MLL-r in these patients, congenital leukemia is very rare with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-expresses MLL-ENL and LIN28B, we investigate the impact of LIN28B on AML. LIN28B impedes the progression of MLL-r AML through compromised leukemia-initiating cell activity and suppression of MYB signaling. Mechanistically, LIN28B directly binds to MYBBP1A mRNA, resulting in elevated protein levels of this MYB co-repressor. Functionally, overexpression of MYBBP1A phenocopies the tumor-suppressor effects of LIN28B, while its perturbation omits it. Thereby, we propose that developmentally restricted expression of LIN28B provides a layer of protection against MYB-dependent AML.

Noncanonical β-catenin interactions promote leukemia-initiating activity in early T-cell acute lymphoblastic leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography–mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin– and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

3077 – FUNCTIONAL AND MOLECULAR IDENTIFICATION OF A FETAL-ASSOCIATED TUMOR SUPPRESSOR AXIS: ABROGATION OF MLL-ENL DRIVEN ACUTE MYELOID LEUKEMIA

MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r have been suggested to be sufficient to drive transformation. However, despite the prenatal origin of MLL-r, the incidence of congenital leukemia is very low with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific RNA binding protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-express MLL-ENL and LIN28B, we investigated the impact of LIN28B on AML. We show that LIN28B impedes the progression of MLL-r AML through a compromised leukemia initiating cell activity and a suppression of MYB signaling. Mechanistically, we found that LIN28B directly bound to MYBBP1A mRNA, resulting in elevated protein levels of this co-repressor of the transcription factor MYB. Overexpression of MYBBP1A in MLL-ENL driven leukemogenesis largely phenocopied the tumor suppressor effects of LIN28B. Thereby, our work proposes that the developmentally-restricted expression of LIN28B during fetal development provides a layer of protection against MYB-dependent AML. MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r have been suggested to be sufficient to drive transformation. However, despite the prenatal origin of MLL-r, the incidence of congenital leukemia is very low with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific RNA binding protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-express MLL-ENL and LIN28B, we investigated the impact of LIN28B on AML. We show that LIN28B impedes the progression of MLL-r AML through a compromised leukemia initiating cell activity and a suppression of MYB signaling. Mechanistically, we found that LIN28B directly bound to MYBBP1A mRNA, resulting in elevated protein levels of this co-repressor of the transcription factor MYB. Overexpression of MYBBP1A in MLL-ENL driven leukemogenesis largely phenocopied the tumor suppressor effects of LIN28B. Thereby, our work proposes that the developmentally-restricted expression of LIN28B during fetal development provides a layer of protection against MYB-dependent AML.

PHF6 and JAK3 Mutations Cooperatively Drive T-Cell Acute Lymphoblastic Leukemia Progression

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent hematologic malignancies resulted from gene mutations and/or genomic rearrangements that occur in T cell progenitors. The 5-year survival rate of T-ALL patients is less than 50%. Much efforts have been dedicated to decipher the molecular events underlying TALL transformation, with the goals to identify specific therapeutic targets and develop new and more effective drugs. As a member of JAK kinase family, JAK3 mutations can be identified in 16.1% of T-ALL cases, and JAK3 M511I mutation is the most common one within all JAK3 mutations. Activating JAK3 M511I mutation induced a lympho-proliferative disorder, that followed by a T-ALL-like disease. PHF6 mutation is one of the most common co-existing gene mutations with JAK3 in T-ALL patients. Co-mutation events of JAK3 and PHF6 account for 1.89%-10.0% in T-ALL cases. However, the role(s) of PHF6 and JAK3 co-mutations in tumorigenesis is unknown.Here in this study, we first analyzed the genetic data of 449 T-ALL cases from multiple clinical centers in which JAK/STAT mutations is about 21% of the total cases. Interestingly, we found that PHF6 mutations were significantly associated with JAK3 mutations in these T-ALL cases (P<0.05), and the JAK3 and PHF6 co-mutation occurred in 7.2% of the cohort. Significantly, the average survival time of PHF6 and JAK/STAT co-mutated group was much shorter than that of the single JAK/STAT mutated group (P<0.05) or none-PHF6/JAK/STAT mutated group (P<0.001). We generated Phf6 KO+JAK3 M511I mice by transplanting JAK3M511I infected Phf6 Lin - cells into wild-type mice. All Phf6 KO+JAK3M511Imice succumbed to leukemia from 74 to 101 days after transplantation with significantly shorter survival time than that of Phf6WT+JAK3M511Imice. The Phf6 KO+JAK3M511I mice showed more aggressive phenotypes of T-ALL than Phf6WT+JAK3M511I mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked increase in leukemia-initiating cell activity in Phf6 KO +JAK3M511Icells when compared with Phf6WT +JAK3M511Icells, supporting a role for loss of Phf6 in promoting leukemia blast self-renewal and proliferation.To investigate the underlying molecular mechanisms of Phf6 in accelerating T-ALL development,we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 in isogenic Phf6WT +JAK3M511I or Phf6KO +JAK3M511IT-ALL cells. Gene set enrichment analysis (GSEA) showed up-regulated cell cycle in Phf6 KO +JAK3 M511I leukemia cells. We then performed Chromatin immunoprecipitation sequencing (ChIP-seq) and found that PHF6 associated with BAI1 gene. qPCR and Western blot showed that the mRNA and protein expression of Bai1/BAI1 were significantly decreased in Phf6KO +JAK3M511Icells in comparison with Phf6 WT+JAK3M511I cells. BAI1 has been reported to prevent MDM2-mediated P53 ubiquitination, and loss of BAI1 reduces P53 level. We found that the protein expression of BAI1 and P53 was decreased, and the ubiquitination of P53 was significantly increased in PHF6 knockdown (KD) MOLT-4 cells (T-ALL) when compared with the control cells. BAI1 overexpression in PHF6 KD MOLT-4 cells significantly increased P53 expression in PHF6 KD+BAI1 OE MOLT-4 cells when compared with that of PHF6 KD cells. It suggested that Phf6 deficiency increased BAI1-mediated P53 degradation. To confirm this, we treated Phf6 KO + JAK3M511I mice with a JAK3 inhibitor (ruxolitinib) in combination with a MDM2 inhibitor (idasanutlin), and found a synergistic response of the treatment within a significant attenuation in leukemia burden in vivo.In conclusion, we found that PHF6 mutation frequently co-existed with JAK3 mutation in T-ALLs, and together they can drive an aggressive leukemia in mice model. Notably, PHF6 deficiency promoted JAK3-induced T-ALL progression by inhibiting BAI1-MDM2-P53 signaling pathway that is independent of JAK3-STAT signaling pathway. We further demonstrated that combination therapy by tofacitinib and idasanutlin can reduce the Phf6 KO and JAK3 M511I leukemia burden in vivo. Our study suggested that combinational usage of JAK3 inhibitors and MDM2 inhibitors may increase the drug benefit for T-ALL patients with PHF6 and JAK3 co-mutations. DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide and Interferon-Alpha Eradicate Adult T Cell Leukemia Initiating Cells through PML/p53 Senescence and Activation of Innate Immunity

Adult T cell leukemia (ATL) is an aggressive chemo-resistant T cell malignancy secondary to chronic HTLV-I infection. The viral oncoprotein Tax initiates ATL in transgenic mice. We previously showed that arsenic trioxide (As) and interferon-α (IFN) combination, known to trigger proteasome mediated Tax degradation and apoptosis in ATL cells, cures murine ATL through clearance of leukemia initiating cells (LIC). We also showed that the triple combination of As, IFN and zidovudine is highly effective in the treatment of chronic ATL patients, inducing a shift from an immunosuppressive Treg/Th2 profile at diagnosis (high IL-10; low IL2; low IFN-γ) towards a Th-1 immunocompetent profile (high IFN-γ; high IL2; low IL-10).To dissect the molecular basis of As/IFN-induced ATL LIC eradication, primary ATL SCID mice were treated with As/IFN for 3 days. Spleen derived ATL cells were injected into secondary recipient SCID mice left untreated and sacrificed on a weekly basis. Whereas, secondary mice derived from untreated primaries succumbed from ATL between 3 and 4 weeks after transplantation, we observed a biphasic growth intersected by a spontaneous regression phase in untreated secondary mice transplanted from As/IFN treated primary mice. This process was associated with telomere shortening, DNA damage, activation of the PML/P53 axis, and senescence in tumor cells. Interestingly, injection of spleen derived ATL cells from secondary SCID sacrificed before ATL senescence, into untreated tertiary and quaternary SCID, resulted in normal ATL development, indicating that senescence of ATL cells in secondary SCID mice resulted in a change of the microenvironment that culminated in ATL cell apoptosis and loss of ATL LIC activity. Furthermore, injection of spleen derived ATL cells from untreated secondary SCID sacrificed before or after ATL senescence, into untreated tertiary NSG mice, resulted in normal ATL development. These NSG mice, contrary to SCID mice, lack natural killer (NK) cells and have an impaired macrophage activity. In agreement with these results, injection of spleen derived ATL cells from treated primary SCID into untreated secondary NSG mice, resulted in normal ATL development. RT-PCR and ELISA analysis of sorted CD25 positive (ATL) or negative (microenvironment) cells revealed treatment-induced upregulation of MIP1, RANTES, and IFN-γ in the CD25 negative fraction in untreated secondary SCID, following ATL senescence, and concomitant with apoptosis and loss of LIC activity. Conversely, and as previously seen in treated ATL patients, IL-10 expression was rapidly and dramatically downregulated in the CD25 positive fraction in untreated secondary SCID mice. Addition of the proteasome inhibitor PS-341 to AS/IFN treatment of primary mice reverted all above results, presumably indicating the role of As/IFN-induced Tax degradation in the observed phenotypes.Collectively, these results demonstrate that As/IFN-induced, PML/p53 dependent, senescence of ATL cells resulted in activation of the innate immunity that culminated in apoptosis and loss of ATL LIC activity. The striking similarity of these results with patients' data suggests a critical role for innate immunity in ATL cure. DisclosuresHermine:Novartis: Research Funding; Hybrigenics: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; INatherys: Equity Ownership, Research Funding.

Clonal Evolution Mechanisms and Collateral Sensitivity to Impdh Inhibitor Therapy in Relapsed Lymphoblastic Leukemia

Improved support and intensified chemotherapy regimens have increased the overall survival rates of newly diagnosed pediatric ALL to over 80%. However the outcomes of patients with relapsed or refractory ALL remain poor, with cure rates of about only 40%. Thus, leukemia relapse and chemotherapy resistance are now the most prominent challenges in the treatment of ALL. Leukemia-initiating cells capable of self-renewal, protective microenvironment safe-haven niches and clonal evolution with acquisition of secondary genetic alterations driving chemotherapy resistance have all been implicated as drivers of ALL disease progression and relapse. In this context, heterozygous activating mutations in the NT5C2 nucleotidase gene are present in about 20% of relapsed pediatric T-cell ALL (T-ALL) cases and 3-10% of relapsed B-precursor ALLs. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during leukemia initiation, disease progression and relapse remain unknown. Using a conditional inducible leukemia model, we demonstrate that expression of Nt5c2 p.R367Q, a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-MP at the cost of impaired leukemia cell growth and leukemia-initiating cell activity. Consistently ultra-deep sequencing with molecular barcodes, allele specific PCR and droplet PCR analyses showed that NT5C2 mutations are typically absent at the time of diagnosis or represent very small clones below the threshold of detection (1:1000). Metabolomic profiling of isogenic Nt5c2 wild type and Nt5c2 p.R367Q mutant lymphoblasts showed that the loss of fitness phenotype of Nt5c2 mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine nucleotide pool. Consequently, blocking guanosine synthesis via inosine-5'-monophosphate dehydrogenase (IMPDH) inhibition induced increased cytotoxicity against NT5C2-mutant leukemia lymphoblasts. Similar results -increased resistance to 6-MP, depleted intracellular nucleoside pool and excess export of purines to the culture media and increased sensitivity to mizoribine- were documented in human ALL cell lines with expression of relapse associated NT5C2 mutations (p.R238W, p.K359Q, p.R367Q and p.D407A). The specificity of these interactions was supported by metabolic rescue of mizoribine-induced cytotoxicity in cultures supplemented with guanosine. Similarly shRNA knockdown of IMPDH2 induced increased cytotoxicity in human cell lines expressing mutant NT5C2. Finally, we verified increased response to mizoribine in relapsed-leukemia derived xenograft cells harboring NT5C2 mutations compared with their corresponding NT5C2 wild type diagnostic-sample derived counterparts. In all, these results identify NT5C2 mutation-associated fitness cost and resistance to chemotherapy as key evolutionary drivers shaping clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL. DisclosuresNo relevant conflicts of interest to declare.

MYC in T-cell acute lymphoblastic leukemia: functional implications and targeted strategies.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that frequently occurs in children and adolescents, which results from the transformation of immature T-cell progenitors. Aberrant cell growth and proliferation of T-ALL lymphoblasts are sustained by activation of strong oncogenic drivers. Mounting evidence highlights the critical role of the NOTCH1-MYC highway toward the initiation and progression of T-ALL. MYC has been emphasized as a primary NOTCH1 transcriptional target impinging in leukemia-initiating cell activity particularly responsible for disease onset and relapse. These findings lay a foundation of T-ALL as an ideal disease model for studying MYC-mediated cancer. The biology of MYC deregulation in T-ALL supports innovative strategies for therapeutic targeting of MYC. To summarize the relevant literature and data in recent years, we here provide a comprehensive overview of the functional importance of MYC in T-ALL development, and the molecular mechanisms underlying MYC deregulation in T-ALL. Finally, we illustrate the innovative MYC-targeted approaches that have been evaluated in pre-clinical models and shown significant efficacy. Given the complexity of T-ALL molecular pathogenesis, we propose that a combination of anti-MYC strategies with conventional chemotherapies or other targeted/immunotherapies may provide the most durable response, especially for those patients with relapsed and refractory T-ALL.

Bone marrow niche ATP levels determine leukemia-initiating cell activity via P2X7 in leukemic models

How particular bone marrow niche factors contribute to the leukemogenic activities of leukemia-initiating cells (LICs) remains largely unknown. Here, we showed that ATP levels were markedly increased in the bone marrow niches of mice with acute myeloid leukemia (AML), and LICs preferentially localized to the endosteal niche with relatively high ATP levels, as indicated by a sensitive ATP indicator. ATP could efficiently induce the influx of ions into LICs in an MLL-AF9-induced murine AML model via the ligand-gated ion channel P2X7. P2x7 deletion led to notably impaired homing and self-renewal capacities of LICs and contributed to an approximately 5-fold decrease in the number of functional LICs but had no effect on normal hematopoiesis. ATP/P2X7 signaling enhanced the calcium flux-mediated phosphorylation of CREB, which further transactivated phosphoglycerate dehydrogenase (Phgdh) expression to maintain serine metabolism and LIC fates. P2X7 knockdown resulted in a markedly extended survival of recipients transplanted with either human AML cell lines or primary leukemia cells. Blockade of ATP/P2X7 signaling could efficiently inhibit leukemogenesis. Here, we provide a perspective for understanding how ATP/P2X7 signaling sustains LIC activities, which may benefit the development of specific strategies for targeting LICs or other types of cancer stem cells.

Loss of interleukin-10 activates innate immunity to eradicate adult T-cell leukemia-initiating cells.

Adult T-cell leukemia/lymphoma (ATL) is associated with chronic human T-cell leukemia virus type 1 infection and carries a poor pr o gnosi s. Arsenic tr ioxide (AS) and inter feron-alpha (IFN) together selectively trigger Tax viral oncoprotein degradation and cure Tax-driven murine ATL. AS/IFN/zidovudine treatment achieves a high response rate in patients with chronic ATL. Interleukin 10 (IL-10) is an immuno-suppressive cytokine whose expression is activated by Tax. Here we show that, in ATL, AS/IFN-induced abrogation of leukemiainitiating cell activity requires IL-10 expression shutoff. Loss of IL-10 secretion drives production of inflammatory cytokines by the microenvironment, followed by innate immunity-mediated clearance of Tax-driven leukemic cells. Accordingly, anti-IL-10 monoclonal antibodies significantly increased the efficiency of AS/IFNtherapy. These results emphasize the sequential targeting of malignant ATL cells and their immune microenvironment in leukemia-initiating cell eradication and provide a strong rationale to test the AS/IFN/anti-IL10 combination in ATL.

Targeting Leukemia-Initiating Cells in Acute Lymphoblastic Leukemia.

The concept that different leukemias are developmentally distinct and, like in normal hematopoiesis, generated by restricted populations of cells named leukemia-initiating cells (LIC), is becoming more established. These cancer stem-like cells have been assumed to have unique properties, including the capability of self-renewing and giving rise to "differentiated" or non-LICs that make up the whole tumor. Cell populations enriched with LIC activity have been characterized in different hematopoietic malignancies, including human acute lymphoblastic leukemia (ALL). Related studies have also demonstrated that LICs are functionally distinct from bulk cells and modulated by distinct molecular signaling pathways and epigenetic mechanisms. Here we review several biological and clinical aspects related to LICs in ALL, including (i) immunophenotypic characterization of LIC-enriched subsets in human and mouse models of ALL, (ii) emerging therapeutics against regulatory signaling pathways involved in LIC progression and maintenance in T- and B-cell leukemias, (iii) novel epigenetic and age-related mechanisms of LIC propagation, and (iv) ongoing efforts in immunotherapy to eradicate LIC-enriched cell subsets in relapsed and refractory ALL cases. Current conventional treatments do not efficiently eliminate LICs. Therefore, innovative therapeutics that exclusively target LICs hold great promise for developing an effective cure for ALL.

Abstract 5412: Xenograft based detection of rare leukemic clones in minimal residual disease negative diagnostic specimens from pediatric patients

Abstract Despite achieving complete remission with negative evaluation for minimal residual disease (MRD-) in the bone marrow, a significant proportion of pediatric leukemia patients experience relapse of disease. This conundrum highlights the limitations of standard methods in detecting extremely rare residual blasts. The heterogenous nature of leukemia also complicates the detection of minor residual subclones that may differ from the dominant clone and thus escape molecular and/or immunophenotypic detection during post-therapy assessments. Novel methods to detect, expand and characterize post-therapy residual disease would potentially allow new prognostic and diagnostic insights into leukemia relapse and its prevention. In the current study, we have engrafted MRD- samples into immune deficient mice in an attempt to use the patient-derived xenograft (PDX) assay to provide a functional readout for clinically undetected residual leukemia-initiating cell (LIC) activity. To date, we have successfully detected residual LICs in clinically MRD- specimens taken from 10 pediatric cases (5 B-ALL and 5 AML). The cases with positive functional LIC detection followed two general scenarios - outgrowth of undetectable marrow disease in the setting of extramedullary relapse, and outgrowth of undetectable marrow disease following either stem cell transplant or chimeric antigen receptor T-cells (CAR-T) therapy. Six bone marrow samples from patients with isolated CNS or extra-medullary relapse were able to establish engraftment despite MRD negativity on clinical testing. Four of the six patients ultimately succumbed to disease progression, and remission was successfully salvaged in the remaining two. Additionally, another four sets of samples acquired after bone marrow transplant or CAR-T therapy established PDX grafts, thereby potentially allowing for prediction of relapse in some patients. 1 AML and 1 ALL patient relapsed with similar kinetics as seen in the mouse. A second PDX-predicted relapse ALL patient succumbed to transplant complications before overt clinical relapse and a second AML patient subsequently relapsed many months after the positive PDX assay. These results highlight the remarkable sensitivity of immune deficient mice to engraft human LICs. Detection of LICs in MRD- BM specimens via PDX formation could aid in teasing apart the various mechanisms for relapse in individual patients. Additionally, these results provide proof of principle for the use of PDX as a surveillance tool for prediction of leukemic relapse and may identify a subset of patients who could benefit from earlier or additional therapy. Citation Format: Mark Wunderlich, Nicole Manning, Eric O'Brien, Christina Sexton, Luke Byerly, John P. Perentesis, Benjamin Mizukawa, James C. Mulloy. Xenograft based detection of rare leukemic clones in minimal residual disease negative diagnostic specimens from pediatric patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5412.

IL-7R is essential for leukemia-initiating cell activity of T-cell acute lymphoblastic leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R–dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.

Antitumor efficacy of arsenic/interferon in preclinical models of chronic myeloid leukemia resistant to tyrosine kinase inhibitors.

Tyrosine kinase inhibitors (TKIs) are the standard treatment for chronic myeloid leukemia (CML). Despite their clinical success, TKIs are faced with challenges such as treatment resistance, which may be driven by kinase domain mutations, and frequent disease relapse upon the cessation of treatment. The combination of arsenic trioxide (ATO) and interferon-α (IFN) was previously demonstrated to inhibit proliferation and induce apoptosis in CML cell lines, prolong the survival of primary wild-type CML mice, and dramatically decrease the activity of leukemia-initiating cells (LICs). The ATO/IFN combination was tested in vitro on imatinib (IMN)-resistant K562-R and Ar230-R cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays were used to evaluate proliferation and apoptosis, respectively. The acridine orange assay was used to assess autophagy, and quantitative reverse transcription-polymerase chain reaction was used to assess the involvement of the hedgehog (Hh) pathway. In vivo, a retroviral transduction/transplantation T315I BCR-ABL CML mouse model was used to assay the effect of the treatment on survival, tumor burden (histopathology and blood counts), and LIC activity (secondary transplantation). In vitro, ATO/IFN synergized to inhibit proliferation and induce apoptosis of IMN-resistant cells with variant modes of resistance. Furthermore, the preclinical effects of ATO/IFN were associated with induction of autophagy along with inhibition of the Hh pathway. Most remarkably, ATO/IFN significantly prolonged the survival of primary T315I-CML mice and displayed a dramatic impairment of disease engraftment in secondary mice, which reflected decreased LIC activity. Collectively, the ATO/IFN strategy has been demonstrated to have the potential to lead to durable remissions in TKI-resistant CML preclinical models and to overcome various TKI-specific mechanisms of resistance.

Phf6 Loss Enhances HSC Self-Renewal Driving Tumor Initiation and Leukemia Stem Cell Activity in T-ALL.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. SIGNIFICANCE: Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305.

Deletion 6q Drives T-cell Leukemia Progression by Ribosome Modulation.

Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression. SIGNIFICANCE: The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome-mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494.

The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.

Preleukemia and Leukemia-Initiating Cell Activity in inv(16) Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a collection of hematologic malignancies with specific driver mutations that direct the pathology of the disease. The understanding of the origin and function of these mutations at early stages of transformation is critical to understand the etiology of the disease and for the design of effective therapies. The chromosome inversion inv(16) is thought to arise as a founding mutation in a hematopoietic stem cell (HSC) to produce preleukemic HSCs (preL-HSCs) with myeloid bias and differentiation block, and predisposed to AML. Studies in mice and human AML cells have established that inv(16) AML follows a clonal evolution model, in which preL-HSCs expressing the fusion protein CBFβ–SMMHC persist asymptomatic in the bone marrow. The emerging leukemia-initiating cells (LICs) are composed by the inv(16) and a heterogeneous set of mutations. In this review, we will discuss the current understanding of inv(16) preleukemia development, and the function of CBFβ–SMMHC related to preleukemia progression and LIC activity. We also discuss important open mechanistic questions in the etiology of inv(16) AML.

Osteoblast Sorting and Intracellular Staining of CXCL12.

Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone osteoblasts offer a simplified and rapid approach to characterize osteoblasts. We describe a modified procedure of isolating mouse bone marrow osteoblastic cells based on cell surface immunophenotyping. The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are expressed by osteoblasts and bone marrow stroma cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al., 2006) and for supporting leukemia initiating cell activity (Pitt et al., 2015). Here we describe the procedure of intracellular staining of CXCL12.

JAM-C Identifies Src Family Kinase-Activated Leukemia-Initiating Cells and Predicts Poor Prognosis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here, we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation was uniquely found within the JAM-C-expressing LSC compartment. Cancer Res; 77(23); 6627-40. ©2017 AACR.