Expansion Of Leukemic Cells Research Articles

A new signaling cascade linking BMP4, BMPR1A, \u0394Np73 and NANOG impacts on stem-like human cell properties and patient outcome

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients’ bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients’ outcome.

FoxN1-dependent thymic epithelial cells promote T-cell leukemia development.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

Aryl hydrocarbon receptor (AHR) is a novel druggable pathway controlling malignant progenitor proliferation in chronic myeloid leukemia (CML).

Aryl Hydrocarbon Receptor (AHR) is an ubiquitous basic helix-loop-helix transcription factor, which is ligand-activated and involved in numerous biological processes including cell division, cell quiescence and inflammation. It has been shown that AHR is involved in normal hematopoietic progenitor proliferation in human cells. In addition, loss of AHR in knockout mice is accompanied by a myeloproliferative syndrome-like disease, suggesting a role of AHR in hematopoietic stem cell (HSC) maintenance. To study the potential role of AHR pathway in CML progenitors and stem cells, we have first evaluated the expression of AHR in UT-7 cell line expressing BCR-ABL. AHR expression was highly reduced in UT-7 cell expressing BCR-ABL as compared to controls. AHR transcript levels, quantified in primary peripheral blood CML cells at diagnosis (n = 31 patients) were found to be significantly reduced compared to healthy controls (n = 15). The use of StemRegenin (SR1), an AHR antagonist, induced a marked expansion of total leukemic cells and leukemic CD34+ cells by about 4- and 10-fold respectively. SR1-treated CML CD34+ cells generated more colony-forming cells and long-term culture initiating cell (LTC-IC)–derived progenitors as compared to non-SR1-treated counterparts. Conversely, treatment of CML CD34+ cells with FICZ, a natural agonist of AHR, induced a 3-fold decrease in the number of CD34+ cells in culture after 7 days. Moreover, a 4-day FICZ treatment was sufficient to significantly reduce the clonogenic potential of CML CD34+ cells and this effect was synergized by Imatinib and Dasatinib treatments. Similarly, a 3-day FICZ treatment contributed to hinder significantly the number of LTC-IC-derived progenitors without synergistic effect with Imatinib. The analysis of molecular circuitry of AHR signaling in CML showed a transcriptional signature in CML derived CD34+ CD38- primitive cells with either low or high levels of AHR, with an upregulation of myeloid genes involved in differentiation in the “AHR low” fraction and an upregulation of genes involved in stem cell maintenance in the “AHR high” fraction. In conclusion, these findings demonstrate for the first time that down-regulation of AHR expression, a major cell cycle regulator, is involved in the myeloproliferative phenotype associated with CML. AHR agonists inhibit clonogenic and LTC-IC-derived progenitor growth and they could be used in leukemic stem cell targeting in CML.

Abstract 3169: Mechanisms of novel cytokine therapy for Ph-like B-cell acute lymphoblastic leukemia with overexpression of CRLF2

Abstract B-cell acute lymphoblastic leukemia with overexpression of CRLF2 (CRLF2 B-ALL) comprises ~50% of Ph-like B-ALL, a leukemia that is associated with poor outcomes, high relapse rates and leukemia health disparities in Hispanic children. CRLF2, together with the IL-7 receptor alpha (IL-7Ra), comprises a receptor complex that is activated by the cytokine, TSLP. Receptor activation induces JAK2/STAT5 and PI3/AKT/mTOR signals that are believed to contribute to survival and proliferation of leukemia cells. To study the role of TSLP in CRLF2 B-ALL, we developed a novel patient-derived xenograft (PDX) model of CRLF2 B-ALL that allows us to vary circulating levels of human TSLP (hTSLP). Primary CRLF2 B-ALL cells injected into PDX mice without hTSLP or with circulating hTSLP levels similar to pediatric leukemia patients (~4-10 pg/ml) showed engraftment and expansion of leukemia cells. In contrast, CRLF2 B-ALL cells were essentially eliminated in PDX with elevated physiologic levels of hTSLP (40-140 pg/mL). We observed these results in 5 independent experiments produced using primary CRLF2 B-ALL cells from two different Hispanic pediatric patients with CRLF2 B-ALL (N= 40 PDX). We hypothesize that the observed antileukemia effects are mediated via TSLP-induced upregulation of the Suppressor of Cytokine Signaling (SOCS) genes. SOCS genes encode a family of proteins (SOCS1-7 and CISH) that regulate cytokine signaling via negative feedback through multiple mechanisms including ubiquitin-mediated cytokine receptor degradation. Consistent with our hypothesis, SOCS1, SOCS2, SOCS3 and CISH mRNA were upregulated in primary CRLF2 B-ALL cells cultured with high-dose hTSLP. To gain mechanistic insights we evaluated the CRLF2 B-ALL cell lines, MUTZ5 and CALL4, following culture with and without hTSLP. Flow cytometry analysis showed that high-dose hTSLP upregulated SOCS1 and SOCS3 proteins in both CRLF2 B-ALL cell lines. We found that CRLF2 B-ALL cells cultured with hTSLP for 3 days showed a dose-dependent loss in the ability to induce STAT5 and S6 phosphorylation following hTSLP stimulation. This loss was correlated with the loss of IL-7Ra, and maintained for 24-48 hours following a pulse of high-dose (but not low-dose) hTSLP. The loss of signaling and surface IL-7Ra could be prolonged if high-dose hTSLP levels were maintained. These data provide evidence that TSLP exerts its antileukemia effects by shutting down CRLF2-mediated signals and suggest that these effects are at least partially mediated by the loss of the IL-7Ra component, and potentially through SOCS family proteins. These studies identify the human TSLP cytokine as a potential biologic therapy to treat CRLF2 B-ALL and reduce cancer health disparities for Hispanic children with CRLF2 B-ALL. (Supported by 1R01CA209829.) Citation Format: Cornelia Stoian, Jacqueline S. Coats, Hossam Alkashgari, Veriah Vidales, Ineavely Baez, Juliette Personius, Hannah Choi, WayAnne Watson, Brandon Ng, Benjamin Becerra, Rishikesh Chavan, Muhammad Kamal, Shadi Farzin Gohar, Sinisa Dovat, Kimberly J. Payne. Mechanisms of novel cytokine therapy for Ph-like B-cell acute lymphoblastic leukemia with overexpression of CRLF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3169.

GATA2 haploinsufficiency accelerates EVI1-driven leukemogenesis

AbstractChromosomal rearrangements between 3q21 and 3q26 induce inappropriate EVI1 expression by recruiting a GATA2-distal hematopoietic enhancer (G2DHE) to the proximity of the EVI1 gene, leading to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The acquisition of G2DHE by the EVI1 gene reciprocally deprives this enhancer of 1 of the 2 GATA2 alleles, resulting in a loss-of-function genetic reduction in GATA2 abundance. Because GATA2 haploinsufficiency is strongly associated with MDS and AML, we asked whether EVI1 misexpression and GATA2 haploinsufficiency both contributed to the observed leukemogenesis by using a 3q21q26 mouse model that recapitulates the G2DHE-driven EVI1 misexpression, but in this case, it was coupled to a Gata2 heterozygous germ line deletion. Of note, the Gata2 heterozygous deletion promoted the EVI1-provoked leukemic transformation, resulting in early onset of leukemia. The 3q21q26 mice suffered from leukemia in which B220+ cells and/or Gr1+ leukemic cells occupied their bone marrows. We found that the B220+Gr1–c-Kit+ population contained leukemia-initiating cells and supplied Gr1+ leukemia cells in the 3q21q26 leukemia. When Gata2 expression levels in the B220+Gr1–c-Kit+ cells were decreased as a result of Gata2 heterozygous deletion or spontaneous phenomenon, myeloid differentiation of the B220+Gr1–c-Kit+ cells was suppressed, and the cells acquired induced proliferation as well as B-lymphoid–primed characteristics. Competitive transplantation analysis revealed that Gata2 heterozygous deletion confers selective advantage to EVI1-expressing leukemia cell expansion in recipient mice. These results demonstrate that both the inappropriate stimulation of EVI1 and the loss of 1 allele equivalent of Gata2 expression contribute to the acceleration of leukemogenesis.

Anti-leukemic activity of microRNA-26a in a chronic lymphocytic leukemia mouse model.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eμ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Abstract 3726: INCB52793 JAK1 inhibitor synergizes with ATRA to inhibit expansion of AML

Abstract Dysregulated JAK/STAT signaling is known to drive myeloproliferative neoplasms, and targeting JAK1 and JAK2 has led to improvement in morbidity and mortality in these diseases. While dose-dependent anemia and thrombocytopenia limit the use of JAK2 inhibition, selectively targeting JAK1 has been explored as a means to suppress inflammation and STAT-associated neoplastogenesis. Recently, INCB52793 was found to be 100-fold selective for JAK1 over JAK2, and it has recently been explored in the clinic in solid tumors and acute myeloid leukemia (AML). In a large high throughput screen, we detected synergistic effects between INCB52793 and all-trans retinoic acid (ATRA) in several non-promyelocytic AML cell lines. In another in vitro assay, human primary AML blasts exposed to INCB52793 exhibited a marked increase in both CD13 and CD86, two markers indicative of cellular differentiation. Given these findings, we tested this combination in an in vivo murine model of AML. Human leukemia cells were injected into the tail vein of sublethally irradiated NSGS mice which were then treated days 7-35 post-transplant with ATRA, INCB52793, ATRA/INCB52793, or vehicle. Weekly monitoring for peripheral human CD45+ cells revealed that the INCB52793/ATRA combination effectively decreased the expansion of leukemic cells. At 35-40 days, significant decreases in tumor burden were seen within the bone marrow (BM) and spleens of INCB62793/ATRA treated mice. Bone marrow and splenic cells were also analyzed by mass cytometry, simultaneously measuring 35 signaling, differentiation, and cell death attributes per-cell. The few remaining human cells in the INCB62793/ATRA combo group synergistically displayed 30-fold decreases in CD38, 8-fold increases in CD34, and attained high levels of p-STAT3 and p-STAT5, potentially implying a resistant progenitor population. Label free proteomics revealed significant fold changes in in vitro INCB52793/ATRA treated cells. Proteins related to cellular differentiation mechanisms, such as SMAD3, BCL11A, RUNX2, HNRNPLL, and SAMHD1, were elevated between 24 to 48 hours post treatment supporting our hypothesis that JAK1 inhibition enhanced ATRA induced differentiation. Targeting retinoic acid receptor and JAK1 together synergistically resulted in the decreased expansion of multiple AML cell lines, and preferential reduction of AML cells from the blood, spleen and bone marrow of treated mice in vivo. Common CD34-CD38+ tumor cells were eliminated, and rare remaining CD34+ AML cells displayed high p-STAT3 and p-STAT5 levels after INCB52793/ATRA therapy. While ATRA is a critical component in the therapy of acute promyelocytic leukemia (M3), it has not been successfully employed in other AML. These preliminary data represent a potential for INCB52793/ATRA therapy in non-M3 AML. Citation Format: Haley E. Ramsey, P. Brent Ferrell, Melissa A. Fischer, Agnieszka E. Gorska, Caroline Maier, Jeremy Norris, Melissa Farrow, Danielle Guiterrez, James Pino, Sandra Zinkel, Carlos Lopez, Holly Koblish, Matthew Stubbs, Peggy Scherle, Jonathan M. Irish, Richard Caprioli, Michael R. Savona. INCB52793 JAK1 inhibitor synergizes with ATRA to inhibit expansion of AML [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 3726. doi:10.1158/1538-7445.AM2017-3726

Reduced hematopoietic stem cell frequency predicts outcome in acute myeloid leukemia.

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse.

Multidisciplinary insight into clonal expansion of HTLV-1\u2013infected cells in adult T-cell leukemia via modeling by deterministic finite automata coupled with high-throughput sequencing

BackgroundClonal expansion of leukemic cells leads to onset of adult T-cell leukemia (ATL), an aggressive lymphoid malignancy with a very poor prognosis. Infection with human T-cell leukemia virus type-1 (HTLV-1) is the direct cause of ATL onset, and integration of HTLV-1 into the human genome is essential for clonal expansion of leukemic cells. Therefore, monitoring clonal expansion of HTLV-1–infected cells via isolation of integration sites assists in analyzing infected individuals from early infection to the final stage of ATL development. However, because of the complex nature of clonal expansion, the underlying mechanisms have yet to be clarified. Combining computational/mathematical modeling with experimental and clinical data of integration site–based clonality analysis derived from next generation sequencing technologies provides an appropriate strategy to achieve a better understanding of ATL development.MethodsAs a comprehensively interdisciplinary project, this study combined three main aspects: wet laboratory experiments, in silico analysis and empirical modeling.ResultsWe analyzed clinical samples from HTLV-1–infected individuals with a broad range of proviral loads using a high-throughput methodology that enables isolation of HTLV-1 integration sites and accurate measurement of the size of infected clones. We categorized clones into four size groups, “very small”, “small”, “big”, and “very big”, based on the patterns of clonal growth and observed clone sizes. We propose an empirical formal model based on deterministic finite state automata (DFA) analysis of real clinical samples to illustrate patterns of clonal expansion.ConclusionsThrough the developed model, we have translated biological data of clonal expansion into the formal language of mathematics and represented the observed clonality data with DFA. Our data suggest that combining experimental data (absolute size of clones) with DFA can describe the clonality status of patients. This kind of modeling provides a basic understanding as well as a unique perspective for clarifying the mechanisms of clonal expansion in ATL.

Molecular Study of NPM1-A (Nucleophosmin1-A) Mutation in Iraqi Adult Acute Myeloid Leukemia Patients; Its Correlation with Clinicopathological Parameters

Acute myeloid leukemia (AML) is a hematological malignancy of myeloid progenitor cells characterized by anomalous proliferation, inhibition of differentiation and expansion of leukemic cells blocked at the early stage of hematopoiesis. The molecular markers have become a smart tool to further division of AML patients into subgroups. Nucleophosmin (NPM1) mutations are found in approximately 30% of adult AML patients and are associated with a favorable outcome when detected in absence of FLT3-ITD mutation. Over 50 molecular NPM1 mutation variants have been identified; the most common one is NPM1-A mutation. The current study aims to detect; the frequency of NPM1-A in Iraqi adult newly diagnosed AML patients using real time – PCR technique and evaluate the relationship of NPM1-A with age, gender, total white blood cell (WBC) count and FAB subtypes of the disease. The frequency of NPM1-A mutation in newly diagnosed AML patients was 18.86%. Age was non-significantly higher in NPM1-A mutated patients than in patients without mutation (P=0.538). The NPM1-A mutation was predominantly seen in male (P= 0.069). The mutation was non-significantly higher among monocytic subtypes (M5+M4) compared with the other subtypes of the disease (P=0.916). In the NPM1 mutated group, the total WBC count was non-significantly higher than the non-mutated group (P=0.302). These findings suggest; Real time-PCR technique using TaqMan probe was very specific molecular test for this mutation. Mutated NPM1-A patients were associated with increased age, a higher total WBC count, male predominance and the monocytic subtypes of the disease.

Upregulation of Mir-99a Is Associated with Poor Prognosis of Acute Myeloid Leukemia and Promotes Myeloid Leukemia Cell Expansion

Acute myeloid leukemia (AML) is a hereditarily and clinically heterogeneous disease driven by a subpopulation of leukemic stem cells (LSCs) that generate the bulk of leukemic cells. LSCs can resist available treatments and result in disease progression and relapse. High number of LSCs or expression of a LSC related gene signature is independently associated with poor prognosis in AML, supporting the notion for LSCs as important targets for therapeutic intervention. It is now evident that miRNAs contribute significantly to the leukemogenic phenotype. To dissect the miRNA expression profile of LSCs, we performed miRNA array analysis in enriched LSCs cells and non-LSCs (leukemic blast cells) from bone marrow (BM) cells of the same AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a were upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients. The cohort were divided into two groups based on the median expression level of miR-99a in LSCs reference to non-LSCs, and correlation analysis indicated that higher expression levels of miR-99a in LSCs were significantly correlated with worse overall survival and event-free survival in these AML patients. Compared with cord blood CD34+ HSPCs, miR-99a was significantly highly expressed in LSCs from AML patients. The expression level of miR-99a was also higher in K562/A02 (multidrug resistant counterpart of K562 cell line) and K562/G01 (Gleevec resistant counterpart of K562 cell line) than the parental K562 cells. These results point to the importance of a novel role of miR-99a in LSC activity and disease progression.By gain-of-function studies via stable lentiviral transduction, we demonstrated that miR-99a functionally regulated the growth and survival of leukemic cells in vitro and in vivo. Enforced miR-99a expression significantly increased the clonogenic capacity in primary AML LSCs. Further studies in myeloid leukemia cell lines revealed that ectopic miR-99a led to accelerated cell-cycle progression and cell proliferation. MiR-99a overexpression (OE) also promotes leukemic cell survival after exposure to chemotherapeutic agents in AML cell lines. In vivo study in a subcutaneous xenograft model showed that a significant lower tumor inhibition rate was observed overtime in the miR-99a/OE cell injected mice compared to the Ctrl cell injected mice when treated with Ara-C. All these results demonstrated that miR-99a, the miRNA enriched from LSCs at relapse stage, plays an important role in myeloid leukemic cell survival and expansion.To explore the mechanism underlying the enhanced proliferation and chemoresistance meditated by miR-99a, we identified differential gene expression profiles by microarray analysis. The results provide a potential regulatory pathway that links the downregulated targets of miR-99a (i.e., PPP3CA) to the dysregulated key genes associated with leukemic cell expansion. Overexpression of miR-99a led to upregulation of CCNE1, and downregulation of CDKN2A which mediate G1 cell cycle arrest by stabilizing the structure of p53 or inducing p53 activation. All these results suggest an important role of miR-99a in cell cycle entry and progression, leading to the accelerated proliferation and overcoming of chemotherapeutic-agents mediated cell cycle arrest.Collectively, our study provides strong evidence that miR-99a plays an important role in altering biological properties, which may lead to the pathogenesis of myeloid leukemia and chemoresistance induction. The levels of miR-99a may be used as a biomarker for the prognosis and progression of AML. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stromal Cells Derived at Diagnosis and Remission Preferentially Support AML Cell Proliferation in Vitro

Introduction: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells characterized by excessive proliferation of early progenitor cells in the bone marrow (BM). The BM niche is a specialized type of microenvironment (ME) that actively supports growth, maturation and maintenance of hematopoietic stem and progenitor cells. They include a complex network of extracellular matrix proteins, soluble growth factors, and cytokines. The information regarding the clinical significance of leukemic microenvironment for AML initiation and propagation is scarce. The aim of the current study was to characterize the AML BM ME and its crosstalk with leukemic progenitor cells and define the role of BM stromal cells (SCs) in supporting leukemic cell growth in active disease as compared to the complete remission (CR) state.Methods: We developed a unique model of patient own stroma (POS) by growing SCs obtained at diagnosis, CR and relapse in a specific medium, including cytokines and proteins derived from human plasma solution. The growth rate and morphological changes of AML SCs were assessed and their ability to support AML cell expansion was studied by monitoring immunophenotyping alterations of CD34 and CD45 expression using FACS analysis. Levels of secreted MCP-1, IL6, GM-CSF, G-CSF and TNF in the co-culture medium were measured using the bead-based immunoassay CBA (BD Biosciences). In addition, real-time PCR (RT-PCR) amplification was performed in the co-culture to quantify changes aiming to assess the expression of specific genes associated with the maintenance of hematopoietic cells, self-renewal, quiescence regulation and mobilization. The following genes were amplified: CXCL12 (SDF1), transforming growth factor beta 1 (TGFβ), secreted phosphoprotein 1 (SPP1) and angiopoietin 1 (ANGP1).Results: The developed POS model allowed revealing high diversity in the morphological appearance of leukemic SCs obtained from different patients and at various disease stages. Patient SCs were found to grow slower than those of healthy volunteers (fig. 1A). Moreover, the growth rate differed between the patients and disease stages, being highest at relapse and lowest at diagnosis (fig. 1A). Levels of SDF-1, TGF-1 and SPP1 were elevated (X50, X50 and X 300 fold, respectively) in CR stroma compared to that of healthy volunteers. Of note, the level of MCP-1 was increased (2200 pg/ml) in the stroma derived at active disease stages (diagnosis and relapse) compared to CR or normal SCs (1050 pg/ml). Additionally, the expansion of CD34+ leukemic progenitor cells was higher in the presence of CR-derived stroma compared to healthy SCs (fig. 1B). In the co-culture, we observed selective ability of POS to support CD34+ leukemic cell expansion, as detected by flow cytometry (1.5 million cells more in POS than in healthy stroma), as well as differential expression of MCP-1 (x3000 pg/ml) and IL-6 (x9000 pg/ml) cytokines in co-cultures of AML cells and POS. These cytokines are known to be involved in the regulation of cell migration and inflammation.Conclusions: AML BM SCs are heterogeneous and differ from healthy BM SCs in their growth rate, morphology, immunophenotype features and the ability to support expansion of early progenitor CD34+ cells. Moreover, even an AML CR-derived stroma, which is considered "healthy", can provide an advantageous ME to leukemogenesis, as evident by increased proliferation of CD34+ leukemic cells in a co-culture with CR SCs compared to healthy SCs. Additionally, POS was found to selectively support the expansion of AML cells of the same patient. These findings, suggesting a cross-talk between leukemic progenitor cells and their specific POS, may lead to better understanding of the mechanisms underlying AML initiation and relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Mechanism By Which Mutant Nucleophosmin (NPM1) Creates Leukemic Self-Renewal Is Readily Reversed

Acute myeloid leukemia (AML) is self-renewal by immature myeloid precursors that fail to differentiate. An influential 'leukemia stem cell' model thus proposes that leukemogenic proteins augment or introduce a stem cell-like self-renewal program into cells, e.g., by upregulating signaling or transcription factors (TF) emblematic of stem cells (e.g., HOX). We investigated how the most recurrently mutated protein in AML, mutant nucleophosmin (mNPM1), causes leukemic cell expansion. The results challenge this model, but most importantly, open the door to rational targeted therapy for mNPM1 AML. One way of examining for stem cell programs in AML cells is to look at expression patterns of master TF that regulate expression of hundreds of genes and dictate cell fates. Of these select TF, the master TF that create hematopoietic stem cells (HLF etc.) are minimally or not expressed. Instead, there are very high levels of the master TF that drive monocyte and granulocyte lineage fates, PU.1 (SPI1) and CEBPA. Clearly, however, the lineage-programs intended by PU.1/CEBPA are inefficiently executed if at all - mNPM1 AML patient bone marrows had 85-97% cells with a granulocyte-monocyte progenitor phenotype, accumulated at the expense of downstream mature cells (Quek et al, JEM 2016). This aggregation at a lineage-committed, intermediate, naturally proliferative level of the hematopoietic hierarchy suggests an alternative model - instead of introducing a poorly-defined stem cell self-renewal program, mutant proteins disable differentiation programs which usually quench MYC-driven proliferation intrinsic to lineage-progenitors. To better understand how mNPM1 interacts with cellular machinery, we used mass-spectrometry to comprehensively document the protein interactions of endogenous NPM1 in AML cell nuclear and cytoplasmic fractions, the first analysis of this kind. Notably, the NPM1 protein interactome was enriched for PU.1. Critically, wild-type (wt) NPM1/PU.1 was in the nucleus of wtNPM1 AML cells, but mNPM1/PU.1 was in the cytoplasm of mNPM1 AML cells. This was evident clearly also by Western blot of cell fractions and by IF microscopy of primary AML cells and cell lines. Is cytoplasmic dis-location of PU.1 sufficient to explain persistent hematopoietic precursor proliferation? We used murine Pu.1 knock-out hematopoietic precursors transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER) to answer this question - Pu.1 relocation from the cytoplasm to the nucleus by tamoxifen triggered monocytic differentiation that terminated proliferation. Moreover, Pu.1-ER cells, like mNPM1 AML cells, highly express Hox genes, rapidly suppressed upon Pu.1 relocation to the nucleus. Thus, Pu.1 dominantly controls Hox and proliferation, as befitting of a master TF, and persistent HOX expression, like persistent progenitor proliferation, can be caused by Pu.1 loss-of-function. Protein macromolecules like NPM1 require transport factors to exit (exportins) the nucleus. A specific exportin, XPO1, was the major exportin found in the NPM1 interactome. XPO1 interactions with transported cargo can be inhibited by the small molecule drug KPT330. KPT330 10-20 nM rapidly re-located mNPM1 and PU.1 to the nucleus, downregulated MYC, upregulated p27/CDKN1B, upregulated monocyte but not granulocyte differentiation markers, induced morphologic changes of monocyte differentiation, and terminated proliferation of mNPM1 AML cells. The same low nanomolar treatment did not induce differentiation of wtNPM1 AML cells (THP1). Moreover, these KPT330 levels are not toxic to normal hematopoiesis (also shown by others). Thus, rather than gain-of-function of elusive stem cell-like self-renewal, the most frequently mutated protein in AML creates self-renewal by disabling a differentiation program that quenches intrinsic MYC-driven proliferation of lineage-progenitors. These observations are a mechanistic rationale to select refractory/relapsed mNPM1 AML patients for treatment with low well-tolerated doses of KPT330, with a defined molecular pharmacodynamic objective of returning PU.1 to the nucleus, to produce cell cycle exits by differentiation rather than p53-mediated apoptosis (to address chemotherapy resistance), to spare precious normal HSC (good therapeutic index), and directly reverse the basis for leukemic self-renewal (proliferation without differentiation). [Display omitted] DisclosuresLandesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

Cytokine-Mediated Inflammatory Pathways Promote Clonal Evolution and Disease Progression in Acute Myeloid Leukemia

Abstract Background: Inflammatory cytokines secreted in the bone marrow microenvironment play important roles in modulating cell survival, proliferation, differentiation, and immune responses in cancer. Perhaps not surprisingly, there is also an association between chronic inflammation and tumor progression. We recently used an ex vivo functional screen of 94 cytokines to show that the pro-inflammatory cytokines IL1α and IL1β promoted the expansion of AML progenitors in 70% (40/60) of primary samples. We therefore hypothesized that inflammatory cytokines are crucial to clonal expansion and disease progression in AML and that therapeutic targeting of these pathways may circumvent disease heterogeneity. Here we provide in vitro and in vivo evidence that IL1-mediated signaling elicits profound expansion of leukemia progenitors in AML patients harboring various genetic mutations and promotes in vivo clonal expansion and disease progression in a murine AML model. Further, these effects are reversed by targeting IL1 signaling. Methods: We validated the role of IL1 signaling using a shRNA approach and in murine competitive repopulation and bone marrow transplantation models. We evaluated the influence of these cytokines on inflammatory markers using immunoblotting, flow cytometry, and Luminex assays, and assessed strategies to target these pathways using small-molecule inhibitors. Results: IL1 stimulation promoted a 3- to 20-fold increase in growth, survival, and clonogenic potential of AML CD34+ cells, while paradoxically suppressing growth of healthy CD34+ cells. To identify the influence of IL1 on in vivo clonal expansion of healthy and leukemic progenitors simultaneously, we established an in vivo murine competitive repopulation study utilizing TET2-null mice. IL1 treatment promoted clonal expansion of TET2-null myeloid cells over wild-type cells during 6 weeks of IL1β treatment. Consistent with this, both flow cytometry analysis and blood differential counts showed an increased percentage of granulocytes and reduced percentage of lymphocytes in IL1-treated mice. In this model, TET2-null monocytes have greater expression of IL1 than wild-type cells, suggesting IL1 promotes clonal growth of TET2-mutated early leukemic progenitors. Similarly, IL1β and IL1 receptors (IL1R1 and IL1RAP) were overexpressed in IL1-sensitive AML bone marrow and peripheral blood samples compared to nonsensitive AML samples and normal samples. Intracellular FACS showed that the majority of IL1β was secreted by monocytes and to some extent by myeloid progenitors. Accordingly, IL1-sensitive AML samples exhibited trends towards monocytic and myelomonocytic clinical features. Reduced survival of AML cells after monocyte depletion was rescued by IL1 treatment, suggesting that IL1 mediates paracrine regulation of AML cell growth. Silencing of the IL1 receptor, IL1R1, reduced the clonogenic potential of AML primary samples and oncogene (AML1-ETO9a, NRASG12D, and MLL-ENL)-transduced mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer than did recipients of wild-type marrow. We also found that IL1β increased phosphorylation of p38MAPK and MK2, as well as secretion of multiple downstream inflammatory cytokines (IL6, IL8, MCP1, MIP1α, and MIP1β) from CD34+ progenitors, in IL1-sensitive AML samples compared to IL1-nonsensitive progenitors. Conversely, treating AML cells with p38MAPK inhibitors such as doramapimod andralimetinib reduced the growth of AML cells by decreasing p38MAPK and MK2 phosphorylation and reducing secretion of inflammatory cytokines from AML progenitors. Clinical and demographic analyses suggest that AML patients are dependent on IL1 signaling irrespective of mutation status and clinical features. Targeting this unifying mechanism of IL1-mediated clonal expansion may thus have application across heterogeneous AML subtypes. Conclusion: We demonstrate that IL1 promotes in vitro and in vivo clonal expansion of leukemic cells and promotes disease progression in AML. As IL1 signaling is active across heterogeneous disease subtypes, AML patients may therefore benefit from drugs targeting IL1/p38MAPK signaling because of their potential to inhibit AML while enhancing normal hematopoiesis, a significant clinical advantage over traditional chemotherapy. Disclosures Agarwal: CTI BioPharma Corp: Research Funding.

Lineage Switch Under Blinatumomab of a Relapsed Common ALL Co-Expressing Myeloid Markers without MLL Rearrangement

Lineage switch under Blinatumomab of a relapsed Common ALL co-expressing myeloid markers without MLL rearrangementAnnabel Zoghbi, Udo zur Stadt, Beate Winkler, Ingo Müller and Gabriele EscherichBackgroundThe bispecific antibody blinatumomab induces T cell-mediated B cell lysis by adhering to CD3 positive cytotoxic T cells and attaching to CD19, a marker which is commonly seen in lymphoblastic malignancies of the B cell lineage. It has been proven to be an effective therapeutic option for adult and pediatric refractory or relapsed B cell precursor acute lymphoblastic leukemia (ALL).Lineage switch from ALL to a CD19 negative myeloid phenotype is well known in patients with mixed lineage leukemia (MLL) gene rearrangements and has been recently described following blinatumomab treatment in an infant with MLL rearrangement as well as for two patients with MLL rearrangement who were treated with CAR-T cells.Case descriptionA nine year old girl with common ALL ( strong positivity for CD 10,19, 24,34,45, 52 and 79a) with the coexpression of myeloid markers (CD13 86% and CD66c 56%) was treated according to CoALL 08-09 high risk protocol and showed a very slow MRD response. Cytogenetic studies showed a hyperdiploid karyotyp without evidence of a MLL, BCR/ABL or TEL/AML rearrangement. Only one month into maintenance therapy she relapsed with the phenotype of the initial blasts. Because of non-response (M3 marrow) after the first two cycles of treatment accorting to the IntReALL protocol with a M3 marrow. The patient became aplastic for 2 months and developed aspergillus pneumonia, which was treated by combination of antifungals and granulocyte infusions. Nevertherless, blinatumomab treatment was initiated. The patient tolerated treatment well and did not develop a cytokine release syndrome, but eventually recovered granulopoiesis under blinatumomab treatment. Leukemic blasts below the 1% border, however, were detectable throughout immunotherapy. In the absence of other opportunities, we planned on a HSCT and in the event of relapse thereafter re-initiation of Blinatumomab in the allogeneic setting with the new immune system developing. Hence, the patient underwent an uneventful MUD-HSCT in March 2016 following conditioning with TBI/VP-16 and serotherapy with alemtuzumab due to CD52 expression of the blasts. On day 29 after transplant, a routine bone marrow aspiration revealed a second molecular relapse at the order of 2 x 10-3with the same immunophenotype. Thus, immunosuppression with cyclosporine A was drastically lowered and a grade II skin GvHD developed, which was controlled by topic treatment, before CsA was discontinued.Blinatumomab was reinitiated on day 49 after SCT. After two and a half weeks on therapy, an increase in LDH and leukocytes was visible. Bone marrow aspiration showed disease progression with 70% blasts, which now were CD19 negative with the immunophenotypical and morphological attributes of myeloid leukemia. Nevertheless, initial MRD markers still were strongly positive, indicating the common precursor with the initial phenotype. Detailed analysis will be presented. Cytogenetic analysis of the relapse samples revealed no changes.With no curative approach remaining, palliative therapy with cytarabine and thioguanine was initiated which could not control the fulminant expansion of leukemic cells, leading to the patient's death within four days from diagnosis.ConclusionCD19 negative relapses following blinatumomab therapy have been perceived in recent treatment studies, while reports on immunologic and morphologic presentation of a myeloid phenotype are scarce. Our patient coexpressed myeloid markers, CD13 and CD66c, from the start, thus, it is possible that subclones who did not carry CD19 had a pronounced selection advantage during blinatumomab treatment, leading to their vast expansion. DisclosuresNo relevant conflicts of interest to declare.

RANK-RANKL Mediated Bone Destruction in B-Cell Acute Lymphoblastic Leukemia

Survival rates for pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL) have improved dramatically over the past 40 years approaching a current long-term survival rate of 85%. However childhood B-ALL patients continue to confront co-morbidities and their long-term consequences. For example, osteopenia and osteoporosis associated fractures are a common complication of pediatric leukemia at diagnosis, during treatment and in long-term B-ALL survivors. The STeroid-associated Osteoporosis in the Pediatric Population (STOPP) study reported that at ALL diagnosis, 16% of children and adolescents present with bone pain, vertebral compression and low vertebral Bone Mineral Density (BMD) scores, with the greatest incidence of vertebral fractures (VF) seen in the first year following diagnosis (J Clin Endocrinol Metab. 2015, 100:3408-17). Glucocorticoid treatment further elevated fracture risk in this population. These data underscore the need to identify molecular mechanism by which leukemic cells contribute to bone loss, and provide targeted therapies to limit these effects.Our laboratory previously showed that Rag2-/- p53-/- Prkdcscid/scid triple mutant (TM) and p53-/- Prkdcscid/scid double mutant (DM) mice develop spontaneous B-ALL, but only TM animals exhibit dissemination of leukemic blasts to the leptomeninges of the CNS, a poor prognosis feature observed in pediatric and adult ALL patients. We observed that TM leukemic mice also displayed fragile vertebral bones. Using comparative transcriptome analysis, we found that RANKL (Receptor Activator of the Nuclear factor-kB Ligand), a Tumor Necrosis Factor (TNF) superfamily member ligand and a key regulator of B cell and osteoclast differentiation, was expressed at greater levels in TM compared to the DM leukemia cells. RANKL binds to its receptor RANK, which is expressed in osteoclast precursor cells. RANK-RANKL interaction induces signaling in the osteoclast precursors and drives their differentiation into mature bone resorbing osteoclasts (Proc. Natl. Acad. Sci. 1999, 96:3540-3545). Upon adoptive leukemia cell transfer into immune deficient mice, RANKL+ TM but not DM cells caused decreased vertebral trabecular bone density in the recipients. Treatment with the recombinant RANKL antagonist protein Osteoprotegerin (OPG-Fc) inhibited the growth and dissemination of RANKL+TM leukemic cells and attenuated bone destruction in the recipient mice. These data suggested that TM mouse leukemia cells cause bone loss in the absence of glucocorticoid or other chemotherapy agents.We then examined the potential role of RANKL in osteoporosis associated with human B-ALL. RANKL mRNA was expressed by a majority of primary human adult and pediatric B-ALL. To determine whether primary patient B-ALL can cause bone loss, we transplanted RANKL+ human B-ALL samples of multiple cytogenetic high-risk subgroups (Complex, hypo-diploid and Mixed Lineage Leukemia (MLL) rearranged) into NOD.SCID.gC-/-(NSG) recipient mice. Micro-CT imaging and bone density measures in the xenotransplant recipients revealed extensive vertebral trabecular bone destruction. Immuno-histological analysis of the human B-ALL recipient mice demonstrated extensive osteoporotic damage of the long bones and marked RANKL protein expression in the long bones of mice harboring extensive human B-ALL cell burden compared to NSG control mice. To determine whether RANKL-RANK interaction was required for the B-ALL mediated bone destruction, cohorts of NSG mice engrafted with human B-ALL were treated with recombinant OPG-Fc compared to a matched Fc control protein. OPG-Fc treatment did not attenuate leukemia cell expansion and bone marrow burden, but despite bulky disease, the treatment conferred robust protection from bone destruction suggesting that RANKL was a critical mediator of this clinical complication.Our data demonstrate a central role of the RANK-RANKL axis in B-ALL-mediated bone disease and identify an actionable therapeutic target to reduce acute and long-term morbidity. Denosumab, an anti-RANKL antibody has been approved for the treatment of bone metastasis by solid tumors and for post-menopausal osteoporosis. Our pre-clinical studies suggest that Denosumab and other agents that inhibit the RANK-RANKL pathway may be efficacious in patients with B-ALL associated bone degeneration. DisclosuresNo relevant conflicts of interest to declare.

Inactivation of KLF4 promotes T-cell acute lymphoblastic leukemia and activates the MAP2K7 pathway.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a high incidence of relapse in pediatric ALL. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate the onset of leukemia and worsen disease progression are largely unknown. Here, we show that the gene encoding the transcription factor KLF4 is inactivated by DNA methylation in children with T-ALL. In mice, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL by enhancing the G1-to-S transition in leukemic cells and promoting the expansion of leukemia-initiating cells. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7. Our results showed that in murine and pediatric T-ALL, loss of KLF4 leads to aberrant activation of MAP2K7 and of the downstream effectors JNK and ATF2. As a proof-of-concept for the development of a targeted therapy, administration of JNK inhibitors reduced the expansion of leukemia cells in cell-based and patient-derived xenograft models. Collectively, these data uncover a novel function for KLF4 in regulating the MAP2K7 pathway in T-ALL cells, which can be targeted to eradicate leukemia-initiating cells in T-ALL patients.

Upregulation of miR-99a is associated with poor prognosis of acute myeloid leukemia and promotes myeloid leukemia cell expansion.

Leukemia stem cells (LSCs) can resist available treatments that results in disease progression and/or relapse. To dissect the microRNA (miRNA) expression signature of relapse in acute myeloid leukemia (AML), miRNA array analysis was performed using enriched LSCs from paired bone marrow samples of an AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a was upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients, and higher expression levels of miR-99a were significantly correlated with worse overall survival and event-free survival in these AML patients. Ectopic expression of miR-99a led to increased colony forming ability and expansion in myeloid leukemia cells after exposure to chemotherapeutic drugs in vitro and in vivo, partially due to overcoming of chemotherapeutic agent-mediated cell cycle arrest. Gene profiling and bioinformatic analyses indicated that ectopic expression of miR-99a significantly upregulated genes that are critical for LSC maintenance, cell cycle, and downstream targets of E2F and MYC. This study suggests that miR-99a has a novel role and potential use as a biomarker in myeloid leukemia progression.

LYN Kinase in the Tumor Microenvironment Is Essential for the Progression of Chronic Lymphocytic Leukemia

Survival of chronic lymphocytic leukemia (CLL) cells strictly depends on the support of an appropriate tumor microenvironment. Here, we demonstrate that LYN kinase is essential for CLL progression. Lyn deficiency results in a significantly reduced CLL burden invivo. Loss of Lyn within leukemic cells reduces B cell receptor (BCR) signaling including BTK phosphorylation, but surprisingly does not affect leukemic cell expansion. Instead, syngeneic CLL transplantation of CLL cells into Lyn- or Btk-deficient recipients results in a strongly delayed leukemic progression and prolonged survival. Moreover, Lyn deficiency in macrophages hinders nursing functions for CLL cells, which is mediated by direct contact rather than secretion of soluble factors. Taken together, LYN and BTK seem essential for the formation of a microenvironment supporting leukemic growth.

Upregulation of long noncoding RNA MIAT in aggressive form of chronic lymphocytic leukemias.

Long noncoding RNAs (lncRNAs) are non-proten-coding transcripts of more than 200 nucleotides generated by RNA polymerase II and their expressions are tightly regulated in cell type specific- and/or cellular differential stage specific- manner. MIAT, originally isolated as a candidate gene for myocardial infarction, encodes lncRNA (termed MIAT). Here, we determined the expression level of MIAT in established leukemia/lymphoma cell lines and found its upregulation in lymphoid but not in myeloid cell lineage with mature B cell phenotype. MIAT expression level was further determined in chronic lymphocytic leukemias (CLL), characterized by expansion of leukemic cells with mature B phenotype, to demonstrate relatively high occurrence of MIAT upregulation in aggressive form of CLL carrying either 17p-deletion, 11q-deletion, or Trisomy 12 over indolent form carrying 13p-deletion. Furthermore, we show that MIAT constitutes a regulatory loop with OCT4 in malignant mature B cell, as was previously reported in mouse pulripotent stem cell, and that both molecules are essential for cell survival.