Expansion Of Immature Myeloid Cells Research Articles

Molecular profiling of acute myeloid leukemia with Runx1-Runx1t1 fusion

ABSTRACT Introduction Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of immature myeloid cells. Among the genetic alterations in AML, the RUNX1- RUNX1T1 fusion, resulting from the t (8; 21) (q22; q22.1) translocation, is common and linked with a favorable prognosis. However, the complete mutational profile contributing to leukemogenesis remains unclear. This study characterizes the mutational profile of the RUNX1-RUNX1T1 fusion in AML using Next-Generation Sequencing (NGS). Methods Twenty-four newly diagnosed AML patients were selected. DNA and RNA were extracted from bone marrow aspirate samples, and libraries were prepared using the Ion AmpliSeq™ Library Kit. Sequencing was performed on the Ion S5™ system, and data were analyzed using Torrent Suite™ and Ion Reporter™ Software. Variants were annotated, and their clinical significance was assessed via ClinVar. In silico prediction tools, including SIFT and PolyPhen-2, were used for functional impact assessment. Results Patients with RUNX1-RUNX1T1 fusion (n = 3) demonstrated higher initial peripheral blood and bone marrow blast counts and a pronounced prevalence of CD56 and CD19 positivity. The average number of mutations in KIT and NF1 was higher in the fusion group (p = 0.021 and p = 0.049, respectively). In total, 228 genetic variants were identified, with most associated with transcription/kinase signaling pathways (36.8%). Epigenetic regulators were the next most common category (21.5%). ClinVar data revealed pathogenic variants predominantly in the KIT gene, including two missense mutations, p.Asp816Val and p.Asp816Ala, at exon 17. Unique variants were identified exclusively in RUNX1-RUNX1T1 patients, including eight non-reported variants. Among these, a TET2 variant (p.Pro1367Leu) was predicted to have deleterious effects based on in silico tools. Conclusion The molecular profiling of RUNX1-RUNX1T1 fusion offers crucial insights into AML’s genetic landscape. Identifying therapeutic targets and novel variants broadens our understanding of mutations, presenting new research opportunities and potential interventions.

Assessment of a novel NRAS in-frame tandem duplication causing a myelodysplastic/myeloproliferative neoplasm

Myelodysplastic/myeloproliferative diseases of childhood cause a relevant disease burden, and many of these diseases may have a fatal course. The use of next-generation sequencing (NGS) has led to the identification of novel genetic variants in patients with these diseases, advancing our understanding of the underlying pathophysiology. However, novel mutations can often only be interpreted as variants of unknown significance (VUS), hindering adequate diagnosis and the use of a targeted therapy. To improve variant interpretation and test targeted therapies in a preclinical setting, we are using a rapid zebrafish embryo model that allows functional evaluation of the novel variant and possible therapeutic approaches within days. Thereby, we accelerate the translation from genetic findings to treatment options. Here, we establish this workflow on a novel in-frame tandem duplication in NRAS (c.192_227dup; p.G75_E76insDS65_G75) identified by Sanger sequencing in a 2.5-year-old patient with an unclassifiable myelodysplastic/myeloproliferative neoplasm (MDS/MPN-U). We show that this variant results in a myeloproliferative phenotype in zebrafish embryos with expansion of immature myeloid cells in the caudal hematopoietic tissue, which can be reversed by MEK inhibition. Thus, we could reclassify the variant from likely pathogenic to pathogenic using the American College of Medical Genetics (ACMG) criteria.

How Induction Cycles before Remission Influence Long-Term Survival in Acute Myeloid Leukemia

Background ： Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by accumulation and expansion of immature myeloid cells in the bone marrow, peripheral blood and other tissues. Despite the progress made in mechanisms and approval of new therapeutics, the clinical outcome of AML remains suboptimal. The achievement of complete remission (CR) after induction chemotherapy is critical because the prognosis of refractory AML was especially unfavorable. However, little is known about the association between remission after first induction therapy and the overall survival (OS) in different subgroups of AML. Study Design and Methods: We retrospectively analyzed patients with newly diagnosed AML from January 2019 to August 2022. The diagnosis and treatment strategies were based on European Leukemia Net (ELN) criteria and National Comprehensive Cancer Network (NCCN) guideline. Composite complete remission (CRc) rate (CR+CR with incomplete recovery [CRi]) were assessed at the completion of induction or re-induction therapy. Minimal residual disease (MRD) was assessed using multiparameter flow cytometry with a minimum sensitivity of 0.1%. OS was measured from the day of diagnosis until death or the last follow-up date. Results : In total, 679 patients were included and the median age was 59.0 (range, 14.0-93.0) years old. There were 56.8% (386/679) of patients who achieved CRc after the induction therapy (group A), 18.1% (123/679) achieved CRc after the re-induction therapy (group B). The median follow-up period was 24.2 months. Even though there was no significant difference between group A and group B on OS, group A exhibited improved OS when censored at allogenic hematopoietic stem cell transplantation (HSCT) compared to that in group B (group A vs group B:3-year OS rates 65.7% vs 51.1%，p=0.004，Figure A). We further explored the effects of remission depth on survival. The results showed that, after induction therapy, patients achieved CR exhibited superior OS compared with that in CRi (CR vs. CRi, 3-year OS rates 69.4% vs 51.6%, p=0.006). Also, compared with these patients who failed to achieved MRD negativity within re-induction therapy, patients achieved MRD negativity within re-induction therapy showed superior OS when censored at HSCT (MRD- vs. MRD+, 3-year OS rates 64.7% vs 34.9%, p=0.040). After the first reduction therapy, patients achieved CRc but MRD positive benefit from HSCT (HSCT vs Non-HSCT, 3-year rates 83.0% vs 57.9%, p=0.017), but in patients with MRD negativity, HSCT did not improve the survival outcomes (HSCT vs Non-HSCT, 3-year rates 76.8% vs 65.7%, p=0.078). The next-generation sequencing (NGS) results were available in 520 patients. Based on the 2022 ELN risk stratification, the 3-year OS rates were 60.3%, 44.2% and 35.0% in favorable, intermediate and adverse group, respectively. Moreover, there were 43, 47, 60 patients in favorable, intermediate, and adverse subgroups underwent HSCT subsequently. In intermediate risk subgroup, patients who failed to achieve CRc after the first induction therapy, HSCT may serve as an effective intervention to prolong survival (3-year rates 100% vs 12.1%, P<0.001,). On the contrary, patients who achieved CRc in induction therapy, the overall survival were not influenced by HSCT status (3-year rates 81.5% vs 31.7%, p=0.153, Figure B). Conclusion: Our finding indicated that remission after the first cycle of induction therapy may be a prognostic factor for OS. Moreover, earlier and deeper remission may lead to improved survival. For those patients in intermediate risk group, who achieved CRc in induction therapy or achieved CRc with MRD negativity within re-induction therapy, HSCT may not influence the survival outcome.

Role of Acute Myeloid Leukemia (AML)-Derived exosomes in tumor progression and survival.

Acute myeloid leukemia (AML) is a quickly aggressive hematopoietic disorder that progress due to the accumulation and clonal expansion of immature myeloid cells. Despite the latest developments in AML treatment, repeated relapses and drug resistance remain one of the major challenges in treatment of leukemia. Currently, it is well known that the components of the tumor microenvironment such as cellular and non-cellular elements play a critical function in treatment failures of AML, also they are most common cause of complications including suppression of hematopoiesis. Exosomes are membrane-bound extracellular vesicles (EVs) that transfer signaling molecules and have attracted a large amount of attention due to their important role in inter-cellular communication in health and disease. Exosomes participate in the survival and chemoresistance of many leukemia through transferring their rich cargos of molecules including miRNAs, growth factors, and cytokines. The key producers of exosomes that mainly participate to AML pathogenesis are bone marrow mesenchymal stem cell (BMSCs) and AML cell themselves. These cells release an enormous number of exosomes that affect several target cells such as natural killer (NK) and hematopoietic stem cells to the development of leukemia proliferation and progression. In the present study, a comprehensive review of the literature has been done to briefly discuss the biology of exosomes and highlight the role of exosomes derived from AML in the progress of acute myeloid leukemia.

Microrna-211 - a Novel Oncogene in Acute Myeloid Leukemia

MicroRNAs are a class of small, non-coding RNA molecules that regulate gene expression by post-transcriptional silencing and regulate many critical cellular processes. In cancer, microRNAs are emerging as important biomarkers of disease and a subset of microRNAs have well-established roles as drivers of cancer development. MicroRNA-211 (miR-211) is an intronic microRNA encoded on human chromosome 15, and deregulated miR-211 expression has been previously demonstrated in melanoma and a range of other cancers. A role for miR-211 in the hematopoietic system, and/or in the development of acute myeloid leukemia (AML) however remains unexplored.Using microRNA expression profiling, across two independent pediatric AML cohorts, we are the first to identify that miR-211 expression is elevated in a subset of patients, from more than 2-fold and up to 50-fold above non-leukemic remission controls. High expression of miR-211 tends to be clustered in patients with cytogenetically normal and M5 AML subtypes. Using hematopoietic reconstitution experiments in mouse models, we show for the first time that enforced expression of miR-211 alone in hematopoietic stem cells is sufficient to drive a partially-penetrant myeloproliferative disease that phenotypically resembles acute myeloid leukemia. Affected mice have an expansion of immature myeloid cells, expressing CD11b and Gr-1 cell surface markers, with monoblastic cellular morphology. This is accompanied by severe anemia and thrombocytopenia, and dissemination of immature hematopoietic cells into the spleen, liver and extramedullary spaces of the bone marrow. We have also explored the potential co-operation between miR-211 and AML oncogenes. Strikingly, preliminary data suggests miR-211 can co-operate with activating RAS mutations to immortalize cells in vitro, and drive AML in vivo .To further explore the molecular mechanisms of miR-211 in AML, we used RNA sequencing and differential expression analysis to compare transcriptional changes induced by miR-211 overexpression in disease mice relative to empty vector controls. We have identified Lin28a as the most downregulated miR-211 gene target. Lin28a is a well-established target of miR-125b in AML and its knockdown leads to an analogous expansion of myeloid cells in vivo as observed in miR-211-dependent AML.Collectively, our results demonstrate a previously unidentified role for miR-211 as an oncogenic microRNA in the development of AML and suggests that miR-211 may potentially drive the development of AML by blocking myeloid differentiation through the repression of Lin28a . DisclosuresNo relevant conflicts of interest to declare.

Exploiting epigenetically mediated changes: Acute myeloid leukemia, leukemia stem cells and the bone marrow microenvironment.

Acute myeloid leukemia (AML) derives from the clonal expansion of immature myeloid cells in the bone marrow, and results in the disruption of normal hematopoiesis and subsequent bone marrow failure. The bone marrow microenvironment (BME) and its immune and other supporting cells are regarded to facilitate the survival, differentiation and proliferation of leukemia stem cells (LSCs), which enables AML cells to persist and expand despite treatment. Recent studies have identified epigenetic modifications among AML cells and BME constituents in AML, and have shown that epigenetic therapy can potentially reprogram these alterations. In this review, we summarize the interactions between the BME and LSCs, and discuss changes in how the BME and immune cells interact with AML cells. After describing the epigenetic modifications seen across chromatin, DNA, the BME, and the immune microenvironment, we explore how demethylating agents may reprogram these pathological interactions, and potentially re-sensitize AML cells to treatment.

3109 - BM-Mscs From Risk-Stratified AML Patients Support Normal Hematopoiesis and are Anti-Inflammatory in in Vivo Preclinical Models of Hematopoietic Reconstitution and Severe Colitis

Acute myeloid leukemia (AML) represents a heterogeneous group of disorders characterized by the rapid expansion of immature myeloid cells (blasts) in the bone marrow (BM). There is wide disease heterogeneity and patient risk-stratification principally relies on cytogenetic-molecular data. BM mesenchymal stem/stromal cells (BMSC) are key components of the hematopoietic niche thought to contribute to leukemia pathogenesis. We recently reported a correlation between behaviors of AML-derived BMSCs (AML-BMSCs) in vitro and overall survival in AML patients. To elaborate on this finding, we investigated the capacity of AML-BMSCs from risk-stratified de novo AML patients to regulate the homeostasis of healthy CD34+ hematopoietic stem/progenitor cells (HSPC), and to exert anti-inflammatory effects in vivo in a pre-clinical model of severe acute colitis. We report that regardless of risk-group, AML-BMSCs support, similar to HD-BMSCs, the survival, proliferation, differentiation and clonogenecity of CD34+ cells in vitro, and the in vivo immune deficient mice repopulating assays. Additionally, AML-BMSC were capable of reversing the inflammatory phenotype in preclinical models of acute severe colitis, showing a greater anti-inflammatory capacity compared to HD-BMSC. Collectively, these data indicate that AML-BMSCs retain the capacity to support healthy HPSC and can suppress inflammation in vivo.

Abstract 3972: Combined inhibition of MCL1 and BCL-2 with AMG 176 and venetoclax induces anti-tumor effects in primary patient samples and models of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy involving the clonal expansion of immature myeloid cells. Dysregulated expression of the BCL-2 family of proteins has been implicated in AML pathogenesis. Specifically, the anti-apoptotic family members MCL1 and BCL-2 have been reported to play key roles in AML survival. The selective inhibition of these two proteins represents an emerging strategy in AML treatment. AMG 176 is a potent and selective MCL1 inhibitor currently being tested in an AML Phase I clinical trial. Here we describe the activity of AMG 176 and AM-8621, a structural analog, as single agents and in combination with the BCL-2 inhibitor venetoclax, in models of AML. AM-8621 and venetoclax were profiled as single agents against a panel of AML cell lines to characterize their dependency on MCL1 and BCL-2 for survival. A wide range of sensitivities to both compounds was observed, with several lines exhibiting dependency on both MCL1 and BCL-2 for survival, suggesting functional redundancy and a requirement for combined inhibition to maximize response. To test this hypothesis, we profiled a subset of cell lines with the combination of AM-8621 and venetoclax. A synergistic interaction was detected in each cell line, highlighting their codependence on MCL1 and BCL-2. We also evaluated the synergistic potential of this combination on primary AML patient samples. Here, freshly purified bone marrow aspirates were treated with equimolar concentrations of AM-8621 and venetoclax and compared against single agents in a flow cytometry based viability assay. Marked improvements in activity and potency were observed with the combination over either agent alone. We also tested the combination of AMG 176 and venetoclax in a MOLM-13 orthotopic xenograft model of AML. Mice were treated twice weekly with AMG 176 (60 mg/kg) and daily with venetoclax (100 mg/kg). While both single agents achieved significant reductions in MOLM-13 tumor burden (69% and 33% reduction in BLI respectively), the combination exhibited complete inhibition of tumor growth (100% reduction in BLI) and achieved tumor regression relative to the first day of dosing. We next characterized the effects of this combination on subsets of hematopoietic cells in vivo. The reduced affinity of AMG 176 for murine MCL1 (200-fold) required the use of a human MCL1 knock-in mouse for these studies. Terminal analysis of mice treated with the combination or AMG 176 alone showed significant decreases in peripheral blood B-cells and monocytes, whereas venetoclax alone exhibited significant reductions in B-cells only. Analysis of spleens revealed greater reductions in both cell types following treatment with the combination compared with either single agent. These data highlight the promise of combined MCL1 and BCL2 inhibition as a novel therapeutic strategy for the treatment of AML. Citation Format: Sean R. Caenepeel, Tao Osgood, Brian Belmontes, Jan Sun, Elaina Cajulis, Andrew Wei, Angela Coxon, Jude Canon, Paul Hughes. Combined inhibition of MCL1 and BCL-2 with AMG 176 and venetoclax induces anti-tumor effects in primary patient samples and models of acute myeloid leukemia [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 3972.

Ips Technology Revealed the Genetic and Functional Diversity Present in a Secondary AML Patient

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell diseases characterized by inefficient hematopoiesis and risk of progression to acute myeloid leukemia with poor prognosis. Although massive parallel sequencing studies have revealed a number of genomic alterations associated with myelodysplastic syndromes (MDS), the relationships between genetic architecture and clonal evolution in MDS remain poorly understood, mainly due to a difficulty in the ex vivo culture of primary MDS cells and a lack of good animal models. Induced pluripotent stem cells (iPSCs) from MDS patients are expected to provide a new platform for elucidation of the pathogenesis of MDS. We used reprogramming technology and transplantation into immunodeficient mice to functionally dissect the genetically defined subclones of secondary acute myeloid leukemia (AML) evolving from MDS (MDS/sAML).We attempted to generate iPSCs from bone marrow and peripheral blood mononuclear cells of a MDS/sAML patient with normal karyotype and multiple FLT3-internal tandem duplication (ITD) mutations, utilizing episomal methods. We successfully established more than 30 iPSC lines from sAML clones (sAML-iPSC) and 4 normal iPSC lines from normal clones at the same time. sAML-iPSC lines displayed characteristic morphology and expressed pluripotent stem cell markers at the levels comparable to those in isogenic normal iPSC lines and ES cell lines. Five randomly selected sAML-iPSC lines formed teratomas. Most of the sAML-iPSC lines retained normal karyotype even after 30 passages. About half of the sAML-iPSC lines harbored one out of four types of FLT3-ITDs identical to the primary sAML cells, reflecting the mosaicism of the FLT3-ITDs in the patient's sAML cells. Whole exome sequencing and SNP-CGH analysis revealed that all sAML-iPSC lines shared the chromosome 11p uniparental disomy (UPD) and 14 somatic single nucleotide variants (SNVs) identical to those of the primary sAML cells, indicating that these iPSC lines are derived from clonal sAML cells. Additionally, most of the sAML-iPSC lines had a frameshift mutation of CEBPA. Unexpectedly, 2 of the sAML-iPSC lines without any FLT3-ITD mutations had a FLT3-D835Y mutation, which was only detected by droplet digital PCR (ddPCR) at extremely low levels in the primary bone marrow sample.Hematopoietic differentiation efficiency of sAML-iPSC lines was comparable to that of isogenic normal iPSC lines. However, CD34+CD38-CD45+lineage- hematopoietic stem and progenitor cells (HSPCs) from sAML-iPSC lines displayed two to three times enhanced colony formation ability in myeloid-conditioning semifluid medium. Colonies formed by sAML-iPSC-derived HSPCs were predominantly composed of immature myeloblasts and could be replated for at least five rounds. Both FLT3-ITD- and FLT3-D835Y-positive sAML-iPSC-derived hematopoietic progenitor cells (HPCs) exhibited cytokine-independent growth but only FLT3-ITD-positive sAML cell lines retained in vitro sensitivity to AC220. To functionally evaluate the subclonal diversity in vivo, we performed xenotransplantation of HPCs from several sAML-iPSC lines into sublethally irradiated NSG mice. HPCs from all tested sAML-iPSC lines engrafted at 13 to 15 weeks after transplant. HPCs from FLT3-ITD-positive sAML-iPSC lines (n=5) displayed a higher ability to engraft and induced more severe sAML-like disease in recipient mice than those from FLT3-wild-type (wt) sAML-iPSC lines (n=4) (median spleen weight, 145.4 mg vs 55.7 mg; P < .05). Survival of mice injected with FLT3-ITD-positive sAML-iPSC-derived HPCs was shorter than those injected with FLT3-wt sAML-iPSC-derived HPCs (194 days vs 361 days; P< .001). To confirm the transformation properties of the FLT3-ITDs in this MDS/sAML patient, we transduced FLT3-wt sAML-iPSC-derived HPCs with retrovirus expressing a FLT3-ITD and transplanted the HPCs into recipient mice. FLT3-ITD-transduced recipients developed an aggressive sAML-like disease in 100% of recipient mice with shorter latency around 6 to 10 weeks, as evidenced by marked splenomegaly and massive expansion of immature myeloid cells in bone marrow and spleen.Our data suggest that this iPSC-based system could be useful for analysing clonal architecture and biological feature of subclones of MDS/sAML to identify candidate genes responsible for initiation and progression of MDS. DisclosuresTakaori-Kondo:Shionogi: Research Funding; Toyama Chemical: Research Funding; Mochida Pharmaceutical: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Alexion Pharmaceuticals: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Yamanaka:iPS Academia Japan: Consultancy.

Abstract 3531: Autophagy facilitates leukemogenesis in a murine model of MLL-AF9-driven AML

Abstract Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of immature myeloid cells. There is an urgent and unmet need for the development of novel therapeutics against AML. Autophagy is a lysosomal catabolic process that has been implicated in leukemia cell survival and chemotherapy response. However, the biological role of autophagy in AML leukemogenesis remains poorly defined. In this study, we genetically ablated ATG5, a gene that is essential for autophagy, in AML cells during leukemogenesis in a murine model of MLL-AF9-driven AML. The homozygous deletion of ATG5 in AML cells significantly delayed MLL-AF9-induced leukemogenesis and progression in vivo. Likewise, the in vitro deletion of ATG5 in MLL-AF9 driven AML cells demonstrated decreased growth as well as reduced colony forming units. Interestingly, ATG5 deficiency demonstrated an altered differentiation hierarchy of MLL-AF9-induced AML cells in vivo that could be linked to decreased proliferation and suppressed leukemogenesis. Overall, this study provides insight at how autophagy can facilitate AML progression. A more comprehensive understanding on autophagy in AML is needed prior to the deployment of autophagy modulators for the treatment of AML. Citation Format: Qiang Liu, Longgui Chen, Hong-Gang Wang. Autophagy facilitates leukemogenesis in a murine model of MLL-AF9-driven AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3531.

Activation of p53 By Novel MDM2 Antagonist RG7388 Overcomes AML Inherent and Acquired Resistance to Bcl-2 Inhibitor ABT-199 (GDC-0199)

Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities.Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014).Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML.Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML.Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s > 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s > 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h.Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems.Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. DisclosuresNichols:Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.

Aberrant Hedgehog Pathway Activity Marks Clinical MDS Progression and Accelerates Leukemic Transformation in Vivo

Introduction:Myelodysplastic Syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell (HSC) disorders with varying clinical outcomes, but prognosis uniformly worsens with transformation to secondary acute myeloid leukemia (AML). Despite recent progress in genomics, the mechanisms responsible for disease progression are not fully understood as most of the somatic mutations defined thus far can be found at early stages of the disease. Previous studies have identified aberrant activation of the Hedgehog (Hh) signaling pathway in a subset of AML patients and the expression of the Hh-regulated transcription factor GLI2 correlated with inferior overall and progression free survival. In solid tumors, Hh pathway activation has been associated with metastatic disease progression, and we examined its role in MDS progression and transformation to AML.Methods/Results: We initially quantified changes in Hh pathway activity in CD34+ cells isolated from serial bone marrow samples collected from MDS patients at the time of diagnosis and following progression to AML. We found that the expression of the Hh target genes GLI1 and PTCH1 was increased in 67% of patients (4/6) suggesting that pathway activation was involved in the development of secondary AML. We also analyzed gene expression in 135 MDS patients and found significantly higher GLI2 expression in high-risk MDS (N=80) compared to low-risk MDS (N=55) (p=0.036). In addition, bone marrow blast percentage was significantly higher in the MDS cohort with higher GLI2 expression (mean±SEM=7.1±0.7%) than with lower expression (5.4±0.5%, p=0.039).In order to mechanistically study the effects of Hh pathway activation on MDS progression, we studied mice expressing the Nup98-HoxD13 (NHD13) fusion gene under the control of the vav promoter that generates progressive cytopenia and, in some animals, progression to AML. We crossed NHD13 mice with mice conditionally expressing the constitutively active mutant of the Hh signal transduction regulator Smoothened (SmoM2) in hematopoietic cells expressing Mx1-Cre. The survival of double transgenic animals (NHD13/SmoM2) was significantly shorter compared to mice expressing NHD13 (median survival of 3 months vs. 12 months, p<0.001, Fig. 1). Similar to previous reports we found that the expression of SmoM2 alone had no significant effect on survival or hematologic phenotype. Compared to NHD13 mice, NHD13/SmoM2 mice had significantly higher peripheral blood WBC (93.9k vs. 5.2k, p<0.001) and splenomegaly (633mg vs. 462mg, p=0.006) at the time of disease progression. Furthermore, an immature population of myeloid cells (Mac1+/Gr1 dim) was widely present in peripheral blood, bone marrow and spleen of NHD13/SmoM2 mice. We also examined mice prior to the onset of disease (4-8 weeks post Smo M2 induction) and found an increased frequency of myeloid cells (Mac1+/Gr1+) within the peripheral blood compared to wild type and NHD13/Smo M2 mice (wild type – 17.2%, NHD13 - 40.5%, NHD13/SmoM2 - 68%, p<0.01).Within the bone marrow, hematopoietic stem/progenitor cells (Lin-Sca1+cKit+) were depleted within both NHD13 and NHD13/SmoM2 mice. To examine the impact of Hh activation on clonogenic growth and self-renewal potential, we plated bone marrow cells in methylcellulose and found that NHD13/SmoM2 cells contained a significantly higher number of CFU compared to NHD13 cells (37 vs. 2, p=0.002) that was sustained with subsequent passages (400 vs. 30, p<0.001). In order to define the compartment enriched for leukemia initiating potential (LIC) we transplanted Lin- and Lin+ leukemic fractions from NHD13/SmoM2 mice into naive recipients and found that Lin+ cells led to death within 3 months suggesting that the Lin+ compartment has acquired self- renewal potential.Conclusions:We found that the Hh signaling pathway may be aberrantly activated in a subset of secondary AML patients progressing from MDS. We also demonstrated that activation of Hh signaling induces fatal AML in a mouse model of MDS characterized by inferior survival and widespread expansion of immature myeloid cells. This disease progression is driven by the acquisition of self-renewal and tumor initiating potential in differentiated Lin+ hematopoietic cells. Our findings suggest that Hh pathway inhibition may be a promising approach for AML arising from MDS. [Display omitted] DisclosuresMaciejewski:Alexion: Speakers Bureau; Celgene: Speakers Bureau.

Myeloid-derived suppressor cells in the development of lung cancer.

Myeloid-derived suppressor cells (MDSC) are widely implicated in immune suppression associated with tumor progression and chronic inflammation. However, very little is known about their possible role in tumor development. Here, we evaluated the role of MDSC in two experimental models of lung cancer: inflammation-associated lung cancer caused by chemical carcinogen urethane in combination with exposure to cigarette smoke; and a transgenic CC10Tg model not associated with inflammation. Exposure of mice to cigarette smoke alone resulted in significant accumulation in various organs of cells with typical MDSC phenotype (Gr-1(+)CD11b(+)). However, these cells lacked immunosuppressive activity and could not be defined as MDSC. When cigarette smoke was combined with a single dose of urethane, it led to the development of tumor lesions in lungs within 4 months. By that time, Gr-1(+)CD11b(+) cells accumulated in the spleen and lung and had potent immunosuppressive activity, and thus could be defined as MDSC. In the CC10Tg model, accumulation of immunosuppressive MDSC was observed only at 4 months of age, after the appearance of tumor lesions in the lungs. Accumulation of MDSC in both models was abrogated in S100A9 knockout mice. This resulted in a dramatic improvement in survival of mice in both models. Thus, cigarette smoke results in the expansion of immature myeloid cells lacking suppressive activity. Accumulation of bona fide MDSC in both models was observed only after the development of tumor lesions. However, MDSC played a major role in tumor progression and survival, which suggests that their targeting may provide clinical benefits in lung cancer.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

Recovery Of Myeloid Derived Suppressor Cell Subsets Following Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that expand during many inflammatory conditions and malignancies. MDSCs may play an important role following allogeneic hematopoietic stem/progenitor cell transplant (HSCT). MDSCs suppress T-cell, B-cell and dendritic cell responses by a number of mechanisms, including promoting regulatory T cell expansion and producing soluble mediators such as Arginase 1 (Arg-1) and iNOS. MDSCs are divided into two subsets: monocytic (M-MDSCs) and granulocytic (G-MDSCs). MDSC morphology and function differ in various tissues under different inflammatory conditions. In a murine asthma model, M-MDSCs inhibit airway inflammation, but the other subset of MDSCs exacerbated airway inflammation. In a sepsis model, MDSCs exaggerated inflammation in the early stage, but suppressed inflammation in the later stage of sepsis. As the early post-transplant period is characterized by the rapid expansion of immature myeloid cells, we postulated this time period may also be a time when MDSCs might play a major role in modulating immune recovery post-transplant, and aid in the development of immune regulatory networks potentially important in the pathophysiology of graft-versus-host disease (GVHD). In nine patients undergoing allogeneic HSCT, peripheral blood was drawn on the day prior to the start of conditioning, days +4-5, +7-9, +14-16, +21-23, +27-29 and +80-100 post HSCT. White blood cells were quantified, red cell depleted using HetaSep (Stem Cell Technologies), then stained with fluorescence-labelled antibodies against CD45, CD15, CD14, HLA-DR, CD33 and CD66b and analyzed by flow cytometry for MDSC subsets. The soluble mediators iNOS and Arg-1were evaluated by intracellular staining for iNOS and Arg-1 and analyzed by flow cytometry. Four of the nine patients developed acute GVHD (II-IV) and/or extensive chronic GVHD. Early recovery of CD33+CD14+HLA-DR-/low M-MDSCs and CD33+CD15+CD66b+ G-MDSCs was seen post-transplant. Compared to healthy donors, the percentage of M- and G-MDSCs was increased by 3 weeks post-transplant. Interestingly, the patients who went on to develop GVHD had lower percentage and number of M-MDSCs, but inversely had higher numbers of G-MDSCs by day +27-29 and day +80-100 post-transplant (Fig. 1). When compared with healthy donors, the expression of Arg-1 in G-MDSCs, a measure of activation of MDSCs, was increased in patients pre- and post-HSCT, especially at day +80-100; while there was no difference seen iNOS expression in G-MDSCs (Fig 2). The expression of Arg-1 and iNOS in M-MDSCs was increased pre-transplant but fell by day +80-100 post HSCT (Fig 2). Taken together, our pilot data indicates that both M- and G- MDSCs recover early post HSCT and may contribute to the pathophysiology of GVHD. Patients with lower numbers of M-MDSCs and higher numbers of G-MDSCs at earlier time points post-transplant might be at greater risk for developing GVHD. Disclosures: No relevant conflicts of interest to declare.

PhotoImmunoNanoTherapy Reveals an Anticancer Role for Sphingosine Kinase 2 and Dihydrosphingosine-1-Phosphate

Tumor-associated inflammation mediates the development of a systemic immunosuppressive milieu that is a major obstacle to effective treatment of cancer. Inflammation has been shown to promote the systemic expansion of immature myeloid cells which have been shown to exert immunosuppressive activity in laboratory models of cancer as well as cancer patients. Consequentially, significant effort is underway toward the development of therapies that decrease tumor-associated inflammation and immunosuppressive cells. The current study demonstrated that a previously described deep tissue imaging modality, which utilized indocyanine green-loaded calcium phosphosilicate nanoparticles (ICG-CPSNPs), could be utilized as an immunoregulatory agent. The theranostic application of ICG-CPSNPs as photosensitizers for photodynamic therapy was shown to block tumor growth in murine models of breast cancer, pancreatic cancer, and metastatic osteosarcoma by decreasing inflammation-expanded immature myeloid cells. Therefore, this therapeutic modality was termed PhotoImmunoNanoTherapy. As phosphorylated sphingolipid metabolites have been shown to have immunomodulatory roles, it was hypothesized that the reduction of immature myeloid cells by PhotoImmunoNanoTherapy was dependent upon bioactive sphingolipids. Mechanistically, PhotoImmunoNanoTherapy induced a sphingosine kinase 2-dependent increase in sphingosine-1-phosphate and dihydrosphingosine-1-phosphate. Furthermore, dihydrosphingosine-1-phosphate was shown to selectively abrogate myeloid lineage cells while concomitantly allowing the expansion of lymphocytes that exerted an antitumor effect. Collectively, these findings revealed that PhotoImmunoNanoTherapy, utilizing the novel nontoxic theranostic agent ICG-CPSNP, can decrease tumor-associated inflammation and immature myeloid cells in a sphingosine kinase 2-dependent manner. These findings further defined a novel myeloid regulatory role for dihydrosphingosine-1-phosphate. PhotoImmunoNanoTherapy holds the potential to be a revolutionary treatment for cancers with inflammatory and immunosuppressive phenotypes.

Pancreatic Tumors and Immature Immunosuppressive Myeloid Cells in Blood and Spleen: Role of Inhibitory Co-Stimulatory Molecules PDL1 and CTLA4. An In Vivo and In Vitro Study

BackgroundBlood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4.Methodology and Principal Findings103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8+ lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33+CD14−HLA-DR− were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33+CD14+HLA-DR− IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive.ConclusionIn PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

Abstract PL02-01: Inflammation, stem cells and cancer

Abstract Many if not most gastrointestinal cancers arise in the setting of chronic inflammation, and examples include gastroesophageal reflux-induced esophageal adenocarcinoma, H. pylori-associated gastric cancer and colitis-associated colorectal cancer. We have utilized murine models of esophagitis (L2-IL-1β transgenic mice), gastritis (H/K-ATPase-IL-1β and Helicobacter felis infection) and colitis (AOM/DSS) to investigate the role of chronic inflammation in the development of malignancy. In models of chronic gastrointestinal inflammation, there is an early expansion of immature myeloid cells (IMC) followed later by increases in αSMA+ stromal fibroblasts (CAFs). The increase in IMC results from in part from an expansion of splenic progenitors/HSC, while the increased CAFs reflects an increase in mesenchymal stem cells (MSCs) within the preneoplastic tissue. Together, the amplification of myeloid progenitors and MSC-derived stromal cells results in an evolving cancer niche that contributes to expansion of gut progenitors. In the colon, these inflammatory signals can results in crypt fission and monoclonal conversion of intestinal crypt units. Thus, an early amplification of epithelial stem or progenitor cells appears to be a requirement for the development of neoplasia. Eliminating the early cancer niche, which appears to be needed for the initiation of tumors, appears to represent a potential strategy for cancer prevention. Citation Format: Timothy C. Wang. Inflammation, stem cells, and cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PL02-01.

Cutting Edge: IRF8 Regulates Bax Transcription In Vivo in Primary Myeloid Cells

A prominent phenotype of IRF8 knockout (KO) mice is the uncontrolled expansion of immature myeloid cells. The molecular mechanism underlying this myeloproliferative syndrome is still elusive. In this study, we observed that Bax expression level is low in bone marrow preginitor cells and increases dramatically in primary myeloid cells in wt mice. In contrast, Bax expression level remained at a low level in primarymyeloid cells in IRF8 KO mice. However, in vitro IRF8 KO bone marrow-differentiated myeloid cells expressed Bax at a level as high as that in wild type myeloid cells. Furthermore, we demonstrated that IRF8 specifically binds to the Bax promoter region in primary myeloid cells. Functional analysis indicated that IRF8 deficiency results in increased resistance of the primary myeloid cells to Fas-mediated apoptosis. Our findings show that IRF8 directly regulates Bax transcription in vivo, but not in vitro during myeloid cell lineage differentiation.

Mechanism of Myeloid-Derived Suppressor Cell Differentiation in Cancer.

Abstract SCI-31Myeloid-derived suppressor cells (MDSC) represent an intrinsic part of myeloid cell lineage and comprised of myeloid progenitors and precursors of myeloid cells. In healthy host upon generation in bone marrow immature myeloid cells (IMC) quickly differentiate into mature granulocytes, macrophages, or dendritic cells. In a number of pathological conditions (cancer, various infections diseases, sepsis, trauma, bone marrow transplantation, autoimmune abnormalities) increased production of IMC is associated with partial block of their differentiation and most importantly pathological activation of these cells manifests in up-regulation of arginase, inducible nitric oxide synthase (iNOS) and NO production, increased level of reactive oxygen species (ROS). This results in expansion of IMC with immune suppressive activity. Accumulation of MDSC was detected in practically all mouse tumor models and in patients with different types of cancer. In mice, MDSCs are characterized by the co-expression of myeloid lineage differentiation antigen Gr1 and CD11b. In humans, MDSC are currently defined as CD14-CD11b+ cells or more narrowly as cells that express the common myeloid marker CD33 but lack the expression of markers of mature myeloid and lymphoid cells and the MHC class II molecule HLA-DR. Recently, the morphological heterogeneity of these cells in mice was defined more precisely based on the expression of cell-surface markers Ly6G and Ly6C. Granulocytic MDSCs have a CD11b+Ly6G+Ly6Clow phenotype, whereas MDSCs with monocytic morphology are CD11b+Ly6G-Ly6Chigh. These two subpopulations may have different functions. Accumulation of MDSC is caused by different soluble factors. Recent studies have demonstrated that factors implicated in regulating the expansion of MDSCs can be provisionally split into two main groups with partially overlapping activity. The first group includes factors that are produced primarily by tumor cells and promote the expansion of MDSC through myelopoiesis stimulation, which is associated with inhibition of myeloid-cell differentiation. These factors include stem-cell factor (SCF), macrophage colony-stimulating factor (M-CSF), IL-6, granulocyte/macrophage colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF) and others. Signalling pathways triggered by most of these factors in MDSCs converge on signal transducer and activator of transcription 3 (STAT3). One of the potential targets for STAT3 was recently identified as S100A8/A9 proteins. Accumulation of these proteins in myeloid progenitors prevents their differentiation and results in expansion of MDSC. The second group of factors are produced primarily by activated T cells and tumor stroma and directly activate MDSC. These factors, which include IFN gamma, IL-13, IL-4 and TGFβ, among others, activate several different signaling pathways in MDSCs that involve STAT6, STAT1, and NF-kB. Most studies have shown that the immune-suppressive function of MDSCs requires direct cell–cell contact, which indicates that they operate either through cell-surface receptors and/or through the release of short-lived soluble mediators. Currently, a number of clinical trials explores the possibility of regulating immune responses in cancer by depleting ot inactivating MDSC in cancer patients. DisclosuresNo relevant conflicts of interest to declare.