Transformation Of Hematopoietic Stem Cells Research Articles

Abstract 5557: Stem-like T cells maintain latent anticancer activity and underly therapy resistance

Abstract Pre-cancerous cells are normally recognized and eliminated by immune cells. Cancer progresses only when this immunosurveillance system fails. Although immunotherapy has driven the most significant and exciting advances in cancer treatment in modern times, current approaches are running into barriers. An emerging area of interest includes evidence for rare but highly potent stem cell-like T cells. Stem-like T cells combine long-term persistence and high potency with immunological memory. Our recent findings reveal insights into the nature and regulation of stem-like T cells and their potential anticancer activity. Minimal residual disease (MRD) underlies therapeutic resistance, dictates treatment escalation, and predicts patient outcomes. We have shown that the Wnt and PI3K pathways promote the transformation of hematopoietic stem cells (HSCs) into chemoresistant leukemia stem cells (LSCs), which form the root of leukemia initiation and post-treatment recurrence. High-throughput screening revealed that LSCs could be targeted by low dose anthracycline treatment. Mechanistically, low dose, but not high dose anthracyclines indirectly target LSCs by inhibiting their unique properties of immune escape, allowing for their elimination by CD8+ T cells. We employed single-cell genomic and proteomic analysis to investigate immunological changes during development of HSCs, LSCs, and their progeny in response to low dose anthracycline treatment. While leukemia progression results in exhaustion of differentiated CD8+ T cells, stem-like T cells accumulate. However, low dose anthracycline treatment reverses this imbalance. Comparing MRD+ and MRD- patient samples, we found that differential proportions of stem-like T cells persist in the bone marrow of leukemia patients. While T cells of MRD- patients recover following chemotherapy induction, T cell recovery is severely attenuated in MRD+ patients. Furthermore, failure of immunological recovery is driven at least partially by a lack of stem-like T cells in MRD+ leukemia patients. Overall, our studies have revealed that low dose anthracyclines, in contrast to high dose, induce opposing, dichotomous effects on LSCs vs. HSCs and stem-like vs. differentiated T cells. Citation Format: Fang Tao, Sara McElroy, Jacqelyn Nemechek, Irina Pushel, John Szarejko, Santosh Khanal, Todd Bradley, Doug Myers, John M. Perry. Stem-like T cells maintain latent anticancer activity and underly therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5557.

Abstract 280: Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors

Abstract Our purpose is to identify stromal-derived signals required for IDH1/2-mut hematopoietic stem cells (HSCs) to become leukemogenic. Mutations in IDH genes are found in 20% of acute myeloid leukemia (AML) patients and are not transformative. They produce the oncometabolite 2HG which inhibits key epigenetic regulators. We explored whether epigenetic changes in the bone marrow (BM) niche could provide cooperative signals to IDH-mut HSCs leading to their transformation and disease development. We found that 2HG is elevated in the BM plasma of MDS/AML patients with IDH1/2 mutations as compared to IDH-wt patients. Interestingly, 2HG is taken up by BM stromal cells (BMSCs) suggesting it may induce aberrant methylation patterns in them. Indeed, methylome analysis showed hypermethylation in BMSCs of AML patients with IDH1/2 mutations compared to wt. Among the most hypermethylated genes in the IDH-mut stroma are AXIN1, AXIN2 and NDK2, all negative regulators of β-catenin; this pathway activation was previously shown to cause MDS/AML in mice and present in 38% of patients. In agreement with these findings βcat activation in the stroma of IDH-mut vs IDH-wt AML patients showed a strong positive correlation as 50% of IDH-mut patients had activated βcat in their BMSCs. Inquiring whether stroma methylation precedes HSC transformation, we analyzed samples from healthy individuals with IDH-mut clonal hematopoiesis. They exhibited increased global and AXIN1 promoter methylation compared to samples without such clones, but not active βcat in osteoblasts, indicating this might be a later step along with disease development. In fact, transplantation of HSCs from Idh2-mut mice to recipients expressing active βcat in osteoblasts led to increase in blasts and shortened survival compared to wt mice transplanted with Idh2-mut HSCs. As many patients relapse following treatment with IDH inhibitors, we investigated a potential role for βcat signaling in treatment outcome. IDH-mut AML patients who do not respond to treatment with the IDHi present more frequently with activated βcat in osteoblasts compared to responders. Analyzing samples from the same patient pre- and post-treatment with IDHi, we found before treatment most patients were positive for βcat stromal activation, whereas after treatment, all patients with complete response were negative, while patients with progressive disease remained positive. Our model suggests IDH-mut HSCs secrete 2HG inducing epigenetic alterations in the niche, some of which cause activation of the βcat pathway. In turn, this additional signal leads to the transformation of HSCs to dysplastic cells and MDS/AML development. This work uncovers a new mechanism for transformation to MDS/AML and resistance to IDHi treatment. Thus, targeting βcat activation in the stroma, in combination with IDHi, could improve treatment outcome. Citation Format: Paraskevi Vgenopoulou, Luis Flores, Ioanna Mosialou, Junfei Zhao, X. Shawn Liu, Pamela Sung, Virginie Penard-Lacronique, Martin Carroll, Stavroula Kousteni. Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 280.

Inflammation as a driver of hematological malignancies.

Hematopoiesis is a tightly regulated process that produces all adult blood cells and immune cells from multipotent hematopoietic stem cells (HSCs). HSCs usually remain quiescent, and in the presence of external stimuli like infection or inflammation, they undergo division and differentiation as a compensatory mechanism. Normal hematopoiesis is impacted by systemic inflammation, which causes HSCs to transition from quiescence to emergency myelopoiesis. At the molecular level, inflammatory cytokine signaling molecules such as tumor necrosis factor (TNF), interferons, interleukins, and toll-like receptors can all cause HSCs to multiply directly. These cytokines actively encourage HSC activation, proliferation, and differentiation during inflammation, which results in the generation and activation of immune cells required to combat acute injury. The bone marrow niche provides numerous soluble and stromal cell signals, which are essential for maintaining normal homeostasis and output of the bone marrow cells. Inflammatory signals also impact this bone marrow microenvironment called the HSC niche to regulate the inflammatory-induced hematopoiesis. Continuous pro-inflammatory cytokine and chemokine activation can have detrimental effects on the hematopoietic system, which can lead to cancer development, HSC depletion, and bone marrow failure. Reactive oxygen species (ROS), which damage DNA and ultimately lead to the transformation of HSCs into cancerous cells, are produced due to chronic inflammation. The biological elements of the HSC niche produce pro-inflammatory cytokines that cause clonal growth and the development of leukemic stem cells (LSCs) in hematological malignancies. The processes underlying how inflammation affects hematological malignancies are still not fully understood. In this review, we emphasize the effects of inflammation on normal hematopoiesis, the part it plays in the development and progression of hematological malignancies, and potential therapeutic applications for targeting these pathways for therapy in hematological malignancies.

Computer-aided drug discovery of c-Abl kinase inhibitors from plant compounds against chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the neoplastic transformation of hematopoietic stem cells, driven by the Philadelphia (Ph) chromosome resulting from a translocation between chromosomes 9 and 22. This Ph chromosome harbors the breakpoint cluster region (BCR) and the Abelson (ABL) oncogene (BCR-ABL1) which have a constitutive tyrosine kinase activity. However, the tyrosine kinase activity of BCR-ABL1 have been identified as a key player in CML initiation and maintenance through c-Abl kinase. Despite advancements in tyrosine kinase inhibitors, challenges such as efficacy, safety concerns, and recurring drug resistance persist. This study aims to discover potential c-Abl kinase inhibitors from plant compounds with anti-leukemic properties, employing drug-likeness assessment, molecular docking, in silico pharmacokinetics (ADMET) screening, density function theory (DFT), and molecular dynamics simulations (MDS). Out of 58 screened compounds for drug-likeness, 44 were docked against c-Abl kinase. The top hit compound (isovitexin) and nilotinib (control drug) were subjected to rigorous analyses, including ADMET profiling, DFT evaluation, and MDS for 100 ns. Isovitexin demonstrated a notable binding affinity (-15.492 kcal/mol), closely comparable to nilotinib (-16.826 kcal/mol), showcasing a similar binding pose and superior structural stability and reactivity. While these findings suggest isovitexin as a potential c-Abl kinase inhibitor, further validation through urgent in vitro and in vivo experiments is imperative. This research holds promise for providing an alternative avenue to address existing CML treatment and management challenges. Communicated by Ramaswamy H. Sarma

Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation

AbstractIt is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1–/+, Egr1–/+, Csnk1a1/Egr1–/+ mice. A transplantable acute leukemia only developed in the Csnk1a1–/+Trp53–edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The “Myc signature” closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression

Myelodysplastic syndromes (MDS) comprise hematological disorders that originate from the neoplastic transformation of hematopoietic stem cells (HSCs). However, discrimination between HSCs and their neoplastic counterparts in MDS-derived bone marrows (MDS-BMs) remains challenging. We hypothesized that in MDS patients immature CD34+CD38− cells with aberrant expression of immunophenotypic markers reflect neoplastic stem cells and that their frequency predicts leukemic progression. We analyzed samples from 68 MDS patients and 53 controls and discriminated HSCs from immunophenotypic aberrant HSCs (IA-HSCs) expressing membrane aberrancies (CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366 or CD371). One-third of the MDS-BMs (23/68) contained IA-HSCs. The presence of IA-HSCs correlated with perturbed hematopoiesis (disproportionally expanded CD34+ subsets beside cytopenias) and an increased hazard of leukemic progression (HR = 25, 95% CI: 2.9–218) that was independent of conventional risk factors. At 2 years follow-up, the sensitivity and specificity of presence of IA-HSCs for predicting leukemic progression was 83% (95% CI: 36–99%) and 71% (95% CI: 58–81%), respectively. In a selected cohort (n = 10), most MDS-BMs with IA-HSCs showed genomic complexity and high human blast counts following xenotransplantation into immunodeficient mice, contrasting MDS-BMs without IA-HSCs. This study demonstrates that the presence of IA-HSCs within MDS-BMs predicts leukemic progression, indicating the clinical potential of IA-HSCs as a prognostic biomarker.

TIF1β Enhances Self-Renewal Capacity of BCR-ABL Leukemic Stem Cells and Inhibits the Myeloid Differentiation

Chronic myeloid leukemia (CML), characterized by expression of p210-BCR-ABL, is a clonal myeloproliferative disease initiated by malignant transformation of hematopoietic stem cells (HSC). Blastic crisis phase (BC) of CML that shows expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. Transcription intermediary factor TIF1β/TRIM28/KAP1 modulates chromatin structure to repress or activate transcription of target genes. Tif1β is essential for the proliferation and differentiation of normal hematopoietic stem cells, but the role of Tif1β in myeloid leukemia is unclear. Recently, high expression of TIF1β was correlated with poor prognosis in patients with various types of cancer. By analyzing datasets of gene expression in bone marrow (BM) cells in CML patients, we found that TIF1β expression levels were robustly elevated in CML cells at the BC stage. Thus, we hypothesized that TIF1β functions as an oncogene for the development of BCR-ABL leukemia. To determine whether TIF1β promoted the development of BCR-ABL-induced myeloid leukemia in vivo, we generated BCR-ABL;Tif1βf/f;Cre-ERT2 compound mice by crossing Rosa26 locus BCR-ABL conditional knock-in mice and Tif1βf/f;Cre-ERT2 conditional KO mice. We transplanted BM cells isolated from Cre-ERT2 (WT), Tif1βf/f;Cre-ERT2 (Tif1β KO), BCR-ABL;Cre-ERT2 (BCR-ABL), and BCR-ABL;Tif1βf/f;Cre-ERT2 (BCR-ABL;Tif1β KO) mice into lethally irradiated CD45.1+ WT recipient mice, and injected tamoxifen 4 weeks after BM transplantation. While BCR-ABL KI mice showed rapid expansion of leukemic blast cells and died in a short period of time, the deletion of Tif1β gene resulted in a significantly longer survival with reduced tumor burden in BM, liver and spleen in BCR-ABL KI mice. BCR-ABL KI mice mostly developed myeloid leukemia, in contrast, BCR-ABL;Tif1β KO mice dominantly showed B-cell leukemia or bone marrow failure diseases. A subset of BCR-ABL;Tif1β KO mice survived without development of lethal leukemia in the primary and the secondary transplantation settings, indicating that TIF1β maintained the self-renewal capacity of leukemic stem cells (LSCs) but also controlled the myeloid/lymphoid lineage commitment. To elucidate the mechanisms underlying the impaired development of leukemia in BCR-ABL;Tif1βKO mice, we performed RNA sequencing analyses of HSCs isolated from WT, Tif1β KO, BCR-ABL, BCR-ABL;Tif1βKO mice. Gene set enrichment analysis revealed that the loss of Tif1β repressed BCR-ABL-induced activation of proliferative stem cell signatures, but increased expression of myeloid differentiation genes including PU.1 gene in HSCs. We found that BCR-ABL;Tif1βKO HSCs reduced expression levels of Myc and Myc-target genes including Fra1 transcription factor (TF), which was known to promote tumor cell proliferation. Notably, human CML-CP and CML-BC CD34+ cells markedly increased expression levels of MYC and FRA1 genes. To clarify how the Tif1β protein controlled self-renewal of LSCs, we performed ATAC-sequencing in murine HSCs and TIF1β-ChIP-sequencing using BCR-ABL-expressing human leukemia cells. ATAC-seq revealed that BCR-ABL KI HSCs showed enrichments of binding motif of Fra1 in open chromatin and that of PU.1 in closed chromatin, compared to WT HSCs, while BCR-ABL;Tif1βKO HSCs cancelled those enrichments of binding motifs of TFs. TIF1β-ChIP-seq revealed that the TIF1β protein directly bound to a promoter region of the FRA1 gene. TIF1β binding sites were enriched with canonical PU.1- and JUN/FRA1-binding motifs. We also found that treatment with imatinib decreased enrichment of FRA1-binding motif among TIFβ-binding regions in BCR-ABL+ cells, indicating that TIFβ opened chromatin accessibility to activate expression of and transcriptional function of the Fra1 gene in a BCR-ABL dependent manner, but repressed the accessibility of myeloid differentiation regulator TFs. Furthermore, knockdown of Fra1 significantly impaired the proliferation of BCR-ABL stem cells in an in vitro condition. In conclusion, we demonstrate that the Tif1β gene was required for the propagation of BCR-ABL-induced myeloid leukemia stem cell in mice. Tif1β enhanced chromatin accessibility of Fra1 TF to promote transcription of LSC signature genes, but also reduced chromatin accessibility at the PU.1-binding motif, resulting in the enhanced proliferation and limited differentiation of LSCs.

Medium-mass Molecules in Plasma of Patients at Different Stages of Chronic Myeloid Leukemia Progression

Chronic myeloid leukemia (CML) is a clonal tumor disease caused by malignant transformation of hematopoietic stem cells, in which a chimeric BCR-ABL gene is formed as a result of specific translocation of areas between the 9th and 22nd chromosomes. This genetic «defect» ensures the uncontrolled proliferation of myeloid cells. The objective: to analyse the content of medium-mass molecules (MMM) in peripheral venous blood plasma of patients with CML at different stages of the disease and theoretical justification of approaches to the correction of detected changes. Materials and methods. The study enrolled 77 patients with CML, who were divided into three groups according to the stage of disease. The control group included 20 people who were primary donors of the Kyiv City Blood Center. The main parameters characterizing the state of protein and carbohydrate metabolism were analyzed. All patients groups were similar in terms of age, sex, anthropometric parameters, and duration of the disease. All patients during hospitalization were examined by clinical, laboratory, instrumental and special research methods, and if necessary, consulted by specialists. Statistical processing of the results was done by methods of variational statistics, using the Microsoft Excel XP computer program. Results. Today, there are known factors that determine the development of CML, namely: chromosomal abnormalities, ionizing radiation, hematopoietic system diseases, toxic effects of drugs and polluted environment, etc. Changes in the content of MMM in plasma of patients with CML compared to the control group were determined. As the disease progresses, the imbalance of MMM metabolism deepens. The severity of plasma MMM content violations of CML patients is accompanied by endogenous body intoxication, since MMM are universal markers of endogenous metabolic intoxication. Conclusions. Changes in the content of medium-mass molecules in the plasma of patients with chronic myeloid leukemia dictate the need for further in-depth research to determine role in the pathophysiology of the disease for further treatment and reduction of the manifestations of endogenous intoxication. Effective treatment of patients with CML is based on modern diagnostics, timely prescription of drugs and subsequent monitoring of the course of the disease.

AML-060 A Truncated Nucleus Isoform of FGFR1 Drives the Transformation of Leukemia Stem Cells in Leukemogenesis

Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated in the cytoplasm as a result of chromosome translocations. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. In this study, we used the mouse bone marrow transduction and transplantation model to demonstrate the leukemic transformation ability of this tnFGFR1 and identified its critical downstream targets, FLT3 and KIT, in mouse and acute myeloid leukemia (AML) patient samples. Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq were used to examine the related signaling pathways and regulated target genes. We now show that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1 induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expresses tnFGFR1, which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but sensitive to the FLT3 inhibitor quizartinib (AC220), suggesting an alternative approach for the treatment of SCLL. This study demonstrates a novel model for the transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by tnFGFR1. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

A truncated derivative of FGFR1 kinase cooperates with FLT3 and KIT to transform hematopoietic stem cells in syndromic and de novo AML

BackgroundMyeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated.MethodsStem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq was used to examine the related signaling pathways and regulated target genes.ResultsFor the first time, we show that this non-classical truncated form of FGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells in an animal model in vivo. These leukemia cells show a mixed immunophenotype with a B-cell B220 + Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. We further show that de novo human AML also express tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo. Combined treatment with FGFR1 and FLT3 inhibitors provides increased survival compared to FGFR1 inhibition alone.ConclusionsThis study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by the truncated nuclear tnFGFR1 derivative, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with an inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Abstract 5837: A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases

Abstract Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. Stem cell transduction and transplantation in syngeneic mice were used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and the results showed that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. RPPA and RNA-Seq examination of the related signaling pathways and target genes revealed that tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expressed tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo suggesting an alternative approach for treatment of SCLL. This study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases results with the combined effects of direct protein activation by the full-length kinases and gene regulation by the truncated nucleus derivative tnFGFR1, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile. Citation Format: Baohuan Cai, Yun Liu, Yating Chong, Hualei Zhang, Stephanie Mori, Atsuko Matsunaga, Xuexiu Fang, John Cowell, Tianxiang Hu. A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5837.

Dissecting the Genetic and Non-Genetic Heterogeneity of Acute Myeloid Leukemia Using Next-Generation Sequencing and In Vivo Models.

Simple SummaryAcute myeloid leukemia (AML) is an extremely aggressive form of blood cancer with high rates of treatment failure. AML arises from the stepwise acquisition of genetic aberrations and is a highly heterogeneous disorder. Recent research has shown that individual AML samples often contain several clones that are defined by a distinct combination of genetic lesions, epigenetic patterns and cell surface marker expression profiles. A better understanding of the clonal dynamics of AML is required to develop novel treatment strategies against this disease. In this review, we discuss the recent developments that have further deepened our understanding of clonal evolution and heterogeneity in AML.Acute myeloid leukemia (AML) is an extremely aggressive and heterogeneous disorder that results from the transformation of hematopoietic stem cells. Although our understanding of the molecular pathology of AML has greatly improved in the last few decades, the overall and relapse free survival rates among AML patients remain quite poor. This is largely due to evolution of the disease and selection of the fittest, treatment-resistant leukemic clones. There is increasing evidence that most AMLs possess a highly complex clonal architecture and individual leukemias are comprised of genetically, phenotypically and epigenetically distinct clones, which are continually evolving. Advances in sequencing technologies as well as studies using murine AML models have provided further insights into the heterogeneity of leukemias. We will review recent advances in the field of genetic and non-genetic heterogeneity in AML.

3044 – IP-10 LOSS MODULATES ERYTHROID CELL PRODUCTION IN MOUSE MODELS OF MYELOPROLIFERATIVE NEOPLASMS

The malignant transformation of hematopoietic stem cells (HSCs) is a multi-step process. In myeloproliferative neoplasms (MPNs), the JAK2 V617F missense mutation is found in most patients and loss of function TET2 mutations co-occur in approximately 10% of patients. Despite comprehensive genomic profiling, disease heterogeneity exists amongst individuals which cannot be solely resolved by genetic approaches. The microenvironment surrounding HSCs can be diverse and dynamic and deregulated cytokine levels have been implicated in the development of myeloid malignancies. One molecule in particular, IP-10/CXCL10, correlated strongly with disease severity and with JAK2 / TET2 mutational status with highest levels present in the serum of double-mutant patients and in JAK2/TET2 double-mutant mouse models compared to wild-type controls and to other MPN mouse models (e.g., CALRdel52) which recapitulate the MPN phenotype. To test the potential functional role of IP-10 in disease development and maintenance, we generated an allelic series of mutant mouse models carrying mutations in the JAK2, TET2 and IP10 genes. In transplantation experiments, JAK2/TET2 double mutant cells were slowed in their ability to drive an MPN in IP-10-/- animals, but eventually developed a strong disease phenotype, suggesting that the mutant clone itself eventually begins to produce IP-10. In double-mutant JAK2V617F/ V617F IP-10-/- models and triple mutant JAK2V617F/ V617F TET2-/- IP-10-/-, partial amelioration of disease phenotype in some animals as measured by serial monitoring of peripheral blood hemoglobin and hematocrit levels as well as erythroid differentiation assays. Together, these studies suggest that IP-10 may play a role in disease modulation at the red cell progenitor stage and set the stage for future studies exploring its reduction as a potential therapeutic modality in MPNs. The malignant transformation of hematopoietic stem cells (HSCs) is a multi-step process. In myeloproliferative neoplasms (MPNs), the JAK2 V617F missense mutation is found in most patients and loss of function TET2 mutations co-occur in approximately 10% of patients. Despite comprehensive genomic profiling, disease heterogeneity exists amongst individuals which cannot be solely resolved by genetic approaches. The microenvironment surrounding HSCs can be diverse and dynamic and deregulated cytokine levels have been implicated in the development of myeloid malignancies. One molecule in particular, IP-10/CXCL10, correlated strongly with disease severity and with JAK2 / TET2 mutational status with highest levels present in the serum of double-mutant patients and in JAK2/TET2 double-mutant mouse models compared to wild-type controls and to other MPN mouse models (e.g., CALRdel52) which recapitulate the MPN phenotype. To test the potential functional role of IP-10 in disease development and maintenance, we generated an allelic series of mutant mouse models carrying mutations in the JAK2, TET2 and IP10 genes. In transplantation experiments, JAK2/TET2 double mutant cells were slowed in their ability to drive an MPN in IP-10-/- animals, but eventually developed a strong disease phenotype, suggesting that the mutant clone itself eventually begins to produce IP-10. In double-mutant JAK2V617F/ V617F IP-10-/- models and triple mutant JAK2V617F/ V617F TET2-/- IP-10-/-, partial amelioration of disease phenotype in some animals as measured by serial monitoring of peripheral blood hemoglobin and hematocrit levels as well as erythroid differentiation assays. Together, these studies suggest that IP-10 may play a role in disease modulation at the red cell progenitor stage and set the stage for future studies exploring its reduction as a potential therapeutic modality in MPNs.

Phase 1/2 Study Investigating CC-90011, a Potent, Selective, and Reversible Oral Inhibitor of Lysine-Specific Demethylase 1 (LSD1), Plus Concurrent Venetoclax (VEN) and Azacitidine (AZA) in Patients with Acute Myeloid Leukemia (AML)

Phase 1/2 Study Investigating CC-90011, a Potent, Selective, and Reversible Oral Inhibitor of Lysine-Specific Demethylase 1 (LSD1), Plus Concurrent Venetoclax (VEN) and Azacitidine (AZA) in Patients with Acute Myeloid Leukemia (AML)

Mdm2 Maintains Cholesterol Biosynthesis in Hematopoietic Stem/Progenitor Cells Independent of p53

Background: The Mdm2 protein is an E3 ubiquitin ligase that directly interacts with p53 protein leading to its degradation. The expression of MDM2 is controlled by p53 activity through an autoregulatory feedback loop. In addition, a single nucleotide polymorphism (SNP) in the MDM2 promoter modulates its expression and is associated with the risk of cancer. Emerging evidence emphasizes the metabolic activities of MDM2 to be essential for the maintenance of cellular homeostasis. We hypothesized that MDM2 maintains the metabolic homeostasis of hematopoietic stem cells (HSCs) and its downregulation in TP53-mutant leukemias leads to metabolic vulnerabilities independent of p53. Investigation of the metabolic role of MDM2 in hematopoietic stem cells can provide valuable insight into the pathology of TP53 mutant leukemias.Methods: To understand the function of Mdm2 in HSCs, we generated a conditional mouse model driven by Vav-Cre to genetically label and trace the fate of HSCs after heterozygous deletion of Mdm2 in early development and adult bone marrow. We utilized fluorescence microscopy, flow cytometry, apoptosis assays and RNA-seq to functionally characterize the fate of HSCs after heterozygous deletion of Mdm2. Using Trp53 floxed allele and a new Trp53 mutant allele that switches from wildtype to Trp53R172H mutant, we deleted and/or mutated Trp53 gene concomitantly in Mdm2 haplo-insufficient HSCs. Additionally, MDM2 copy number as well as MDM2 SNP309 status were determined in 95 samples from p53 mutant AML patients and 24 controls.Results: Heterozygous deletion of Mdm2 in hematopoietic stem cells (Vav-Cre;mTmG;Mdm2 fl/+) resulted in massive apoptosis of emerging hematopoietic progenitor cells in the aorta-gonad-mesonephros (AGM) region at E11.5. Strikingly, hematopoietic cells residing in fetal liver displayed minimal apoptosis evident by a few TUNEL positive cells. Colony forming assays revealed a myeloid biased hematopoiesis in Mdm2 haplo-insufficient HSCs. Vav-Cre;Mdm2 fl/+ mice displayed a marked reduction in Lin -/CD150 +/c-Kit +/Sca-1 + HSCs cells and significant decrease in peripheral blood counts. Deletion of Trp53 in these mice (Vav-Cre;Trp53 fl/fl;Mdm2 fl/+) resulted in marked decrease in CD19+ B lymphocytes cells whereas the population of CD11b+ myeloid cells did not change. The population of Lin neg-c-Kit + hematopoietic stem/progenitor cells isolated from the bone marrow of Vav-Cre;Mdm2 fl/+ mice displayed marked downregulation of cholesterol biosynthesis and mevalonate pathway (-log2 pvalue=20). Strikingly, 85% of genes involved in cholesterol biosynthesis (29 genes) were downregulated in Vav-Cre;Mdm2 fl/+ mice. Homozygous deletion of Trp53 in Vav-Cre;Mdm2 fl/+ mice did not rescue the metabolic alterations driven by Mdm2 haplo-insufficiency. In addition, the gene signature of oxidative phosphorylation(oxphos), was remarkably upregulated in Vav-Cre;Mdm2 fl/+ mice independent of p53. We further demonstrate that Cre-mediated induction of a Trp53R172 mutation in Mdm2 haplo-insufficient mice resulted in malignant transformation of HSCs leading to acute myeloid leukemia (AML). Of note, mice with homozygote Trp53 mutation and/or deletion without Mdm2- haplo-insufficiency developed lymphoma and not leukemia. In human, MDM2 loss of heterozygosity (MDM2 LOH) in AML was always concomitant with TP53 missense mutations (log2 odds ratio>3, p<.001), and not TP53 deletions or truncations whereas in lymphomas, MDM2 LOH and TP53 mutations were mutually exclusive.Conclusion: Using a genetic model, we have shown that Mdm2 haplo-insufficiency in HSCs leads to apoptosis and clonal selection towards myeloid biased hematopoiesis. Mechanistically, Mdm2 haplo-insufficiency resulted in a metabolic switch from cholesterol biosynthesis to oxphos in HSCs. Notably, this metabolic reprograming is not rescued by deletion of Trp53. However, mutation of Trp53 in Mdm2 haplo-insufficient hematopoietic stem cells resulted in leukemic transformation of HSCs leading to acute myeloid leukemia. Lastly, we demonstrate that MDM2 SNP309 is associated with TP53 mutation in AML and provide clinical evidence that MDM2 loss of heterozygosity is concomitant with TP53 mutations in AML with lower survival compared to TP53 mutant patients with diploid MDM2. Our findings demonstrate a p53-independent role for Mdm2 in metabolic maintenance of hematopoietic stem/progenitor cells. [Display omitted] DisclosuresKhoury: Kiromic: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding. Andreeff: Glycomimetics: Consultancy; Medicxi: Consultancy; Karyopharm: Research Funding; ONO Pharmaceuticals: Research Funding; Senti-Bio: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Syndax: Consultancy; Amgen: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Aptose: Consultancy.

C-MYC Augments the Proliferation and Survival of Hematopoietic Stem Cells and Multipotent Progenitors to Drive Myeloproliferative Neoplasms

Human genomic studies have identified frequent MYC amplification and copy number gains in myeloid malignancies, and previous studies have shown that MYC plays important roles in survival of Myeloproliferative Neoplasms (MPN) and Acute Myeloid Leukemia (AML) cells. Notably, our recent studies have shown that MYC impairs myeloid cell differentiation and promotes proliferation of myeloid progenitors and AML cells by controlling genomic methylation. However, it is unclear if increased levels of MYC in hematopoietic stem cells (HSCs) and myeloid progenitors is sufficient to provoke the development of MPN or AML and, if so, how this occurs.To addresses these questions we generated Mx1-Cre;Rosa26-LSL-MYC transgenic mouse model that inducibly overexpress MYC following polyinosinic:polycytidylic acid (pIpC) injection and Cre-mediated deletion of loxp-stop-loxp cassette. MYC overexpression was confirmed by qRT-PCR and immunoblot. Complete blood counts (CBC) with differential in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice vs. -MYC +/-or -wild type (WT) littermate mice at week 23 revealed worsening anemia (Hb, 9.6 vs. 16.3 vs. 15.5g/dL, p<0.0001), lymphopenia (73.2 vs. 84.3 vs. 84.5%, p<0.0001), and monocytosis (7.4 vs. 1.8 vs. 0.9%, p=0.0097). Also, bone marrow (BM) cells from the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice showed increased monocyte- and granulomonocyte-colony forming potential (CFU-M and CFU-GM), but with limited self-renewal capacity ex vivo (i.e., no CFU after 5 serial plating). Further, inducible MYC overexpression promotes expansion of HSCs (Lin -Sca-1 +cKit + [LSK]), multipotent progenitors (MPPs; LSK CD48 +CD150 -), common myeloid progenitors (CMPs; Lin -Sca1 -cKit +), granulocyte-monocyte progenitors (GMPs; Lin -Sca-1 -cKit +CD34 +FCγR +), and Gr-1/CD11b+ mature myeloid cells, with concomitant reduction of B220+ or CD3+ cells in the BM and spleen. In addition, MYC overexpression provokes splenomegaly (565 vs. 150 vs. 100mg at week 18~22, p<0.0001), extramedullary hematopoiesis with markedly atypical megakaryopoiesis and myeloid preponderance akin to MPN that reduces overall survival (median OS, 157 days vs. not reached vs. not reached, p<0.0001). Collectively, these findings suggest MYC confers enhanced proliferation and survival properties to HSCs and MPPs leading to MPN-like disease.We have shown MYC oncogenic functions in AML cells requires its suppression of TFEB, an mTORC1 regulated bHLH-LZ transcription factor, and that TFEB functions as a tumor suppressor by inducing IDH1/2-TET2 signaling, thus promoting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) conversion in key genes that drive myeloid differentiation and cell death. Similarly, inducible overexpression of MYC in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice significantly reduces the expression of Tfeb, Idh1 and Idh2, and 5hmC levels in both c-Kit + and Cd11b + BM cells. Further, 4-OHT-mediated silencing of Myc in ex vivo cultured BM cells from the Rosa26-CreER T2+/-;Myc fl/fl mice impairs myeloid cell proliferation and robustly induces the expression of Tfeb, Idh1, and Idh2 as well as levels of 5hmC. Finally, inducible TFEB expression in normal 32D.3 myeloid progenitor cells impairs cell proliferation and upregulates 5hmC levels, and these responses are partially reversed by treatment with 2-hydroxyglutarate, an oncometabolite that inhibits 5mC-to-5hmC conversion. Collectively, these findings suggest that the MYC-TFEB-IDH1/2 epigenetic circuit plays a pivotal role in promoting myeloid proliferation to drive the malignant transformation of HSCs to the MPN. DisclosuresKuykendall: Pharmaessentia: Honoraria; Abbvie: Honoraria; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Blueprint: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria, Speakers Bureau. Komrokji: Agios: Honoraria, Speakers Bureau; Acceleron: Honoraria; Geron: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau.

ZNF521 Enhances MLL-AF9-Dependent Hematopoietic Stem Cell Transformation in Acute Myeloid Leukemias by Altering the Gene Expression Landscape.

Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34+ cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34+ cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones.

RUNX3 Promotes the Development of MDS/MPN Overlap Syndrome Via Enhancing Expression of Myc in the Absence of Tet2

The RUNX transcription factors have a critical role in normal and malignant hematopoiesis including myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX3 is expressed in various hematopoietic cells, and the reduced expression of RUNX3 due to promoter DNA hyper-methylation is seen in patients with acute lymphoid leukemia, indicating a tumor suppressive role of RUNX3. The loss of Runx3 promotes the development of MPN-like disease in mice. However, loss-of-function mutations and/or a deletion of RUNX3 are rarely observed in patients with myeloid malignancies. In fact, given that enhanced levels of RUNX3 expression are found in a part of patients with AML and MDS, RUNX3 may play an oncogenic role for myeloid malignancies.To determine how RUNX3 over-expression promotes the development of myeloid malignancies, we generated a mouse model of RUNX3 -induced MDS/MPN by utilizing Tet2 -deficient hematopoietic stem cells (HSCs). Since TET2 loss-of-function mutations initiate the transformation of HSCs, we harvested bone marrow CD150+CD34-LSK HSCs from tamoxifen-treated Cre-ERT2 (WT) and Cre-ERT2;Tet2Δ/Δ (Tet2 KO) mice, and transduced expression of 3xFLAG-tagged RUNX3 by using a retrovirus vector in vitro. Then, we transplanted them into lethally-irradiated Ly5.1+ recipient mice together with wild-type competitor cells. We observed that Tet2 KO mice died dominantly due to MPN-like disease, whereas RUNX3 -expressing Tet2 wild-type (RUNX3/WT) HSCs neither competed wild-type bone marrow cells nor developed hematological malignancies in mice by 12 months post transplantation. In contrast, RUNX3 -expressing Tet2 KO (RUNX3/Tet2 KO) HSCs preferentially generated myeloid cells, but not lymphoid cells, and subsequently competed wild-type cells in the PB and the BM. We found that 7 out of 18 RUNX3/Tet2 KO mice developed lethal MDS/MPN diseases despite a longer latency. At the time of sacrifice, RUNX3-Tet2 KO mice showed severe pancytopenia accompanied with dyplastic blood cells including pseudo-Pelger neutrophils and micro megakaryocytes. In addition, these moribund mice significantly enlarged spleen due to the expansion of myeloid cells. FACS analysis demonstrated that the expansion of LSK hematopoietic stem/progenitor cells (HSPCs) in the BM and the spleen, consistent with the features of extramedullary hematopoiesis. Thus, RUNX3/Tet2 KO mice recapitulated the phenotypic features of human MDS/MPN overlap syndrome, indicating that RUNX3 over-expression is prerequisite to the development of MDS/MPN in the absence of Tet2, although it has an adverse effect on the function of Tet2 -present HSCs, reminiscent with a tumor suppressive function of RUNX3 as seen in Runx3 KO mice.To understand the underlying molecular mechanism in the pathogenesis of RUNX3 -induced MDS/MPN, we performed gene expression analysis in HSPCs isolated from WT, RUNX3/WT, Tet2 KO and RUNX3/Tet2 KO mice at pre-disease and MDS/MPN stages. Gene Set Enrichment Analysis (GSEA) demonstrated that RUNX3/Tet2 KO MDS/MPN cells significantly enhanced expression of c-Myc target genes, compared to either WT cells or RUNX3/Tet2 KO cells at the pre-disease stage. While RUNX3/Tet2 KO cells showed negative enrichments of differentiation-related pathway genes, compared to WT cells, we found that those MDS/MPN cells enhanced expression of cell cycle, ribosome, and mitochondria-related biological pathways, in which c-Myc may regulate target genes and contribute to the transformation. In order to determine whether RUNX3 directly activates expression of c-Myc, we performed ChIP-sequencing by using an anti-FLAG antibody in RUNX3/Tet2 KO bone marrow cells, and found that c-Myc 3' distal enhancer regions showed conserved binding sites for Runx transcription factors. In addition, we found that RUNX3 -expressing human AML cells showed higher sensitivity to JQ1, a BRD4 inhibitor, following the significant reduction of both RUNX3 and C-MYC expression. The growth inhibition by JQ1 was rescued by ectopic expression of RUNX3 .In conclusion, RUNX3 and c-Myc collaborated to activate the function of oncogenic enhancers, in which Tet2 deficiency may alter the epigenetic status for allowing the bindings of these transcription factors, to an extent not seen in RUNX3-expressing alone. We demonstrated that RUNX3 functions as either an oncogene or a tumor suppressor for myeloid malignancies in the context dependent manner. DisclosuresHarada:Celgene: Research Funding; NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding.

Association of SDF-1 Gene Polymorphism with Increased Risk of Acute Myeloid Leukemia Patients.

Background:Acute myeloid leukemia (AML) is a heterogenous group of disorders that emerge from the malignant transformation of hematopoietic stem cells. Chemokine stromal cell-derived factor 1(SDF-1) and its receptor CXC receptor 4 (CXCR4) has an essential role in dissemination of blast cells. Study aimed to detect CXCR4 expression and the SDF-1 (rs1801157) gene polymorphisms and correlate them with prognosis and outcome in AML patients. Subjects and Methods:The study was conducted on 60 de-novo AML patients, and 60 healthy controls. SDF-1 (rs1801157) gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and CXCR4 expression was done using flow cytometry analysis. Results:SDF-1 dominant model (AG+AA) had higher risk AML (p 0.002). CXCR4 positive cases were associated significantly with toxic manifestations (p 0.019), lower CR rates (p 0.004), and unfavorable cytogenetics (p 0.027). Multivariate analysis showed that combined CXCR4positive with dominant SDF-1 considered as independent prognostic factor for shorter overall survival (OS) in AML patients (p 0.031). Conclusion:SDF-1 dominant model had a higher risk to develop AML, and CXCR4 positive expression predicts poor prognosis in AML patients and it could represent a targeted therapy in AML. In addition, CXCR4 could be easily integrated into the initial routine diagnostic work up of AML.

Activating Mutation of SHP2 Establishes a Tumorigenic Phonotype Through Cell-Autonomous and Non-Cell-Autonomous Mechanisms.

Gain-of-function mutation of SHP2 is a central regulator in tumorigenesis and cancer progression through cell-autonomous mechanisms. Activating mutation of SHP2 in microenvironment was identified to promote cancerous transformation of hematopoietic stem cell in non-autonomous mechanisms. It is interesting to see whether therapies directed against SHP2 in tumor or microenvironmental cells augment antitumor efficacy. In this review, we summarized different types of gain-of-function SHP2 mutations from a human disease. In general, gain-of-function mutations destroy the auto-inhibition state from wild-type SHP2, leading to consistency activation of SHP2. We illustrated how somatic or germline mutation of SHP2 plays an oncogenic role in tumorigenesis, stemness maintenance, invasion, etc. Moreover, the small-molecule SHP2 inhibitors are considered as a potential strategy for enhancing the efficacy of antitumor immunotherapy and chemotherapy. We also discussed the interconnection between phase separation and activating mutation of SHP2 in drug resistance of antitumor therapy.