Leukemic Cells In Peripheral Blood Research Articles

Computational modeling reveals key factors driving treatment-free remission in chronic myeloid leukemia patients

Patients with chronic myeloid leukemia (CML) who receive tyrosine kinase inhibitors (TKIs) have been known to achieve treatment-free remission (TFR) upon discontinuing treatment. However, the underlying mechanisms of this phenomenon remain incompletely understood. This study aims to elucidate the mechanism of TFR in CML patients, focusing on the feedback interaction between leukemia stem cells and the bone marrow microenvironment. We have developed a mathematical model to explore the interplay between leukemia stem cells and the bone marrow microenvironment, allowing for the simulation of CML progression dynamics. Our proposed model reveals a dichotomous response following TKI discontinuation, with two distinct patient groups emerging: one prone to early molecular relapse and the other capable of achieving long-term TFR after treatment cessation. This finding aligns with clinical observations and underscores the essential role of feedback interaction between leukemic cells and the tumor microenvironment in sustaining TFR. Notably, we have shown that the ratio of leukemia cells in peripheral blood (PBLC) and the tumor microenvironment (TME) index can be a valuable predictive tool for identifying patients likely to achieve TFR after discontinuing treatment. This study provides fresh insights into the mechanism of TFR in CML patients and underscores the significance of microenvironmental control in achieving TFR.

Resveratrol Inhibits T-acute Lymphoblastic Leukemia in Mice by Regulating Notch1 Signaling Pathway

To observe the effect of resveratrol (Res) on T-acute lymphoblastic leukemia (T-ALL) mice, and further explore its mechanism on Notch1 signaling pathway. Twenty-five 6-8 weeks old female C57BL/6 mice were randomly divided into control group, T-ALL group and Res group. Res group was further divided into low-Res, middle-Res and high-Res group. The percentage of leukemia cells in peripheral blood and spleen cell suspension were detected by flow cytometry and Wright-Giemsa staining, pathological morphology of spleen and bone marrow tissues were observed by HE staining, the expression levels of Notch1, Hes-1, c-Myc, miR-19b and PTEN mRNA in spleen tissue were detected by RT-qPCR, and the protein levels of Notch1, Hes-1, c-Myc, p-PTEN and PTEN were detected by Western blot. Compared with control group, the leukemia cells in peripheral blood of mice in T-ALL group were markedly increased, accompanied by diffuse infiltration of leukemia cells in spleen and bone marrow tissues, the mRNA levels of Notch1, Hes-1, c-Myc, miR-19b and the protein levels of Notch1, Hes-1, c-Myc were increased (P <0.01), while the expression of PTEN mRNA and protein were significantly decreased in the spleen tissue of T-ALL mice (P <0.01). The above indicators in the H-Res group were reversed compared with T-ALL group after administration of resveratrol. Resveratrol may play a role in anti T-ALL by inhibiting Notch1 signaling pathway in mice.

Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications

Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.

Regulatory T cells contribute to the immunosuppressive phenotype of neutrophils in a mouse model of chronic lymphocytic leukemia

BackgroundImpaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL.MethodsIn this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg.ResultsThe obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the “healthy” phenotype of neutrophils.ConclusionsAltogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.

Correlation between D-Dimer/Fibrinogen Ratio and Bleeding in Patients with Newly Diagnosed Acute Promyelocytic Leukemia

To further explore the better indicators for predicting the degree of bleeding associated with newly diagnosed acute promyelocytic leukemia (APL). A total of 131 patients with newly diagnosed APL were classified according to WHO bleeding scales before treatment and divided into two groups: scales 0, 1 and 2 were included in no severe bleeding group, scales 3 and 4 were included in severe bleeding group. The information of the patients were collected, including sex, age, hemoglobin (Hb), white blood cell (WBC) count and platelet (PLT) count, peripheral blood lymphocyte percentage (LYMPH%), peripheral blood monocyte percentage (MONO%), percentage of leukemic cells in pripheral blood and bone marrow, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) levels, D-dimer (D-D), D-dimer/fibrinogen ratio (DFR). Among 131 patients, 110 were classified as no severe bleeding, and 21 were severe bleeding. The results of univariate analysis showed that patients with severe bleeding had significantly higher percentage of leukemic cells in pripheral blood, WBC, D-D, and DFR, as well as longer PT and lower LYMPH%, compared to those with no severe bleeding. Multivariate analysis revealed that DFR (OR =1.054, 95%CI : 1.024-1.084, P < 0.001) and percentage of peripheral blood leukemic cells (OR=1.026, 95%CI: 1.002-1.051, P =0.033) were independent risk factors for severe bleeding. The area under ROC curve (AUC) of peripheral blood leukemic cells, D-D and DFR were 0.748, 0.736 and 0.809, respectively. There was no statistical difference between the peripheral blood leukemic cells and D-D in diagnostic efficacy (P =0.8708). Compared with D-D, DFR had a higher predictive value (P =0.0302). The optimal cut-off value of DFR was 16.50, with a sensitivity of 90.5% and a specificity of 70.0%. DFR has a significant advantage in predicting the degree of bleeding associated with newly diagnosed APL. The greater the DFR value, the heavier the degree of bleeding. The risk of severe or fatal bleeding increases when DFR is greater than 16.50.

Construction of CD19 targeted dual- and enhanced dual-antibodies and their efficiency in the treatment of B cell malignancy

BackgroundT cell-redirecting bispecific antibodies establish a connection between endogenous T cells and tumor cells, activating T cells function to eliminate tumor cells without ex vivo genetic alteration or manipulation. Here, we developed a novel dual-specific antibody (DuAb) and an enhanced DuAb (EDuAb) with different stimulation signal to activate T cells, and evaluated their impact on the treatment of acute lymphoblastic leukemia (ALL).MethodsThe expression plasmids of the DuAb and EDuAb containing CD80 molecule were constructed by cloning heavy chain and light chain variable fragments from anti-human CD19 (HI19a) and CD3 (HIT3a) monoclonal antibody hybridomas, respectively. The activation and the anti-tumor efficacy of human T cells mediated by DuAb and EDuAb were evaluated in vitro. B-cell ALL xenograft NSG mouse model was established to investigate the therapeutic effect in vivo.ResultsEDuAb promoted the optimal expansion of primary human T cells with low expression of inhibitory markers in vitro than DuAb did. Both DuAb and EDuAb showed a similar capability in inducing healthy donor T cells to specifically eliminate B-ALL cell lines and primary blasts from patients. The similar ability was also observed in the patient-derived T cells. In vivo study showed that both DuAb and EDuAb significantly alleviated tumor burden and extended survival of B-ALL xenograft NSG mice. The median survival of PBS, DuAb and EDuAb treatment groups were 27, 38 and 45 days, respectively. The phenotype of T cells and cytokine release in peripheral blood (PB) of B-ALL xenograft NSG mice on day 24 were analyzed as well. The results showed that the proportion of CD8+ T cells and cytokine levels, including IL-2, IFN-γ and TNF-α, were higher in the EDuAb group than that of DuAb. Moreover, both DuAb and EDuAb significantly decreased the residual leukemia cells in PB of B-ALL xenograft NSG mice.ConclusionsBoth DuAb and EDuAb showed great potential as novel treatments for B-ALL in clinical applications. However, compared to DuAb, EDuAb showed a significant advantage in promoting the proliferation and survival of T cells. Furthermore, EDuAb showed a better promising effect on eliminating tumor cells and extending survival in vivo, which provides new insights for the development of new multi-specific antibodies.

Abstract 1353: CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow

Abstract Tumors emerge and evolve in complex and dynamic microenvironments that influence their growth, invasion, and metastasis. In the tumor microenvironment, the immune system plays a critical role in the prevention and elimination of tumors. Despite immune surveillance, tumors still manage to develop through evolution facilitating the establishment of an immune suppressive microenvironment. There is emerging evidence showing that the antitumor immunity of immune effector cells can be obstructed by immune suppressive cell populations such as MDSCs or Tregs. Hematologic malignancies associated with FGFR1 translocations, also known as Stem Cell Leukemia/Lymphoma (SCLL) syndrome, show a complex presentation including myeloproliferative neoplasm, AML, and T- or B-cell lineage lymphoblastic leukemia/lymphoma, providing an ideal, homogeneous model to investigate mechanisms underlying leukemogenesis. Our recent investigation in mouse models of SCLL revealed that mice xenografted with SCLL cells showed increased CD11b+Ly6CHigh M-MDSC and CD11b+Ly6CInt PMN-MDSC levels in the peripheral blood (PB) during leukemia development, accompanied by significantly decreased levels of T cells. To further confirm this MDSC-mediated suppression of T cell function, MDSC depletion experiments were performed in the SCLL cell engraftment model using either the anti-Ly6G antibody (for PMN-MDSCs) or the anti-Gr-1 (aka Ly6G/Ly6C; for total MDSCs) antibody. In both cases, significantly increased levels of CD4+ and CD8+ cells were observed, along with significantly prolonged mouse survival. These observations were consistent in multiple SCLL cell line models, supporting the idea that SCLL cells promote the accumulation of MDSCs, which in turn suppresses the antitumor activity of cytotoxic T cells, allowing SCLL progression. To further characterize these leukemia cell induced MDSCs, CyTOF analysis was performed with the PBMC and bone marrow (BM) samples from leukemic and naïve mice. In peripheral blood leukemia cell induced M-MDSCs also express macrophage markers, including F4-80 and CD64, implying a potential overlap of MDSC and tumor-associated macrophage (TAM). These TAMs also express high CD71 and CD184. Most importantly, we can also identify a CD64/CD71/CD184 expressing cluster in the BM samples. Different from the PB, these cells in the BM express stem cell marker CD117, suggesting there are potential progenitor cells of TAMs. Taken together, our studies demonstrated that SCLL cells establish a tumor favorable microenvironment by reshaping hematopoiesis toward MDSC generation in the BM. A comprehensive understanding of these immune modulation processes holds the promise of improving patient outcomes by reversing the generation of MDSC/TAM in the BM and overturning the establishment of the immunosuppressive microenvironment at the outset. Citation Format: Xiaocui Lu, Ting Zhang, Atsuko Matsunaga, Xuexiu Fang, Stephanie F. Mori, John Cowell, Tianxiang Hu. CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1353.

Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice

The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

DEK-NUP214 Acute Myeloid Leukemia Depends on CRM1 In Vivo

Introduction: Acute myeloid leukaemia (AML) with the translocation DEK-NUP214 (t(6;9)(p23;q34)) is a rare subtype present in 1-2% of AML patients. This translocation is associated with poor clinical outcome, resistance to chemotherapy and remains poorly characterized. Among all nucleoporins, NUP214 has the highest affinity for CRM1, a major exportin protein. We hypothesized that inhibition of CRM1 would be an effective treatment against the aberrant activity of DEK-NUP214. We assessed the leukemia cell line FKH-1, which has a DEK-NUP214 translocation as its sole mutation. In parallel, we established a patient-derived xenograft (PDX) model with the intent to further characterize the disease and assess the efficacy of CRM1 inhibitor KPT-8602/eltanexor. Methods: Cell viability, induction of apoptosis, cell cycle and proliferation were measured at 24h, 72h and 96h for FKH-1 cells after single treatment with eltanexor and cytarabine. Changes in gene expression were assessed by real time PCR. Confocal immunofluorescence microscopy was used to study the localization of target proteins. Leukemic blasts from five DEK-NUP214 positive patients were injected intravenously into irradiated NSGS mice. Engraftment of human cells in peripheral blood was monitored monthly by flow cytometry, and engrafting cells were serially transplanted. The DEK-NUP214 fusion transcript was confirmed by Sanger sequencing and molecular aberrations were evaluated with a custom myeloid sequencing panel. Five weeks after transplantation, treatment with eltanexor or vehicle (CTL) was initiated in 5 mice per group at 12.5 mg/kg (for 12 weeks at 5 days/week, and 4 weeks at 4 days/week). Results: Eltanexor treated FKH-1 cells showed an increase in apoptotic cells in a dose-dependent manner (42.4% at 100 nM vs 23.4% with CTL, p=0.0008), a decrease in cell proliferation, a reduction in overall cell viability, and downregulation of the fusion gene as well as EYA3, PRDM2 and SESN1, which are putative target genes of DEK-NUP214. Although cytarabine treatment induced apoptosis in FKH-1 cells, the downstream targets were not affected, suggesting an on-target effect of eltanexor. Eltanexor inhibited cell cycle (proportion of cells in S phase for eltanexor 1.02% at 400nM vs 7.51% for CTL, p=0.018) and disrupted the co-localization of CRM1 and DEK-NUP214 in the nucleus. In addition, eltanexor reduced the formation of DEK-NUP214 nuclear bodies and reduced protein expression of CRM1 in whole cell lysates. In the first PDX model (PDX1), primary and secondary transplantations showed a mean of 12.5 and 43.9% of CD45+CD33+ cells in peripheral blood at 20-24 weeks. PDX1 mice had high white blood cell counts, low platelets and hemoglobin at time of sacrifice, as well as increased spleen weight and bone marrow blasts, which confirmed the development of AML. The engrafted cells showed a myeloid phenotype with expression of CD38, CD33 and CD14, with higher engraftment in bone marrow than spleen. Mutational analysis showed that the PDX samples retained the same mutations as observed in the patient sample (FLT3-ITD and WT1) besides the DEK-NUP214 fusion transcript. The eltanexor or CTL in vivo treatment started 5 weeks after transplantation similar mean leukemic engraftment between treatment and control groups. After sixteen weeks of treatment and additional 5 weeks of observation, CTL treated mice developed leukemia with 19% engraftment of leukemic cells in peripheral blood, while eltanexor treated mice remained negative, p=0.003. This was accompanied by a decrease in WBC, hemoglobin, and platelet count in CTL treated mice, while the eltanexor treated group retained normal counts. Forty two weeks since transplantation, four vehicle mice have succumbed to disease while eltanexor treated mice remain leukemia free. Gene set enrichment analysis (reactomeDB) from RNAseq data of treated PDX1 cells showed upregulated neutrophil degranulation, signaling by interleukins, specifically IL-10, IL-4, and IL-13, as well as reduced activation of anterior HOX genes in eltanexor vs vehicle treated mice. Conclusion: DEK-NUP214 positive FKH-1 cells are sensitive to CRM1 inhibition by eltanexor, which downregulates putative gene targets of DEK-NUP214 leukemia. We have successfully established a DEK-NUP214 PDX model and show exceptional sensitivity of these cells to eltanexor in vivo.

Risk of Cytokine Release Syndrome with Glofitamab Is Predicted By an Updated Model with a Potential Clinical Application

Background: Cytokine release syndrome (CRS) is a potentially life-threatening toxicity caused by immune activation. CRS can be triggered non-specifically by T-cell engaging therapies. Komanduri et al. (ASH 2021) outlined a model based on eight baseline factors (CRS-risk score; CRS-RS) which allowed accurate classification of risk for Grade ≥2 CRS upon treatment with glofitamab. Glofitamab is a CD20xCD3 T-cell-engaging bispecific monoclonal antibody with a novel 2:1 (CD20:CD3) configuration that has shown promising efficacy with a manageable safety profile in NP30179 (NCT03075696), an ongoing Phase I/II dose-finding study of glofitamab in pts with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL; Dickinson et al. ASCO 2022). While CRS is observed with glofitamab, cases typically occur with Grade 1/2 severity (per ASTCT grading; Lee et al. Biol Blood Marrow Transplant 2019) mostly in the first cycle (C). Here, we introduce a streamlined model based on five baseline factors (CRS-RS.5p) derived from the original CRS-RS, an ongoing non-interventional study (GO43921) to assess the performance of CRS-RS.5p, and the potential application of the risk score in a clinical setting. Methods: In both models, using a weighted sum of baseline clinical, laboratory and imaging parameters, pts are assigned a risk score as a continuous measure for developing CRS after the first dose of glofitamab. Using a defined cut-off, pts are classified as low or high risk of CRS. The original CRS-RS model parameters were: age >64 years at study entry, lactate dehydrogenase pre-anti-CD20 treatment >280 U/L, white blood cell count pre-anti-CD20 treatment >4.5x109 cells/L, Ann Arbor Stage III/IV at study entry, sum of the product of the perpendicular diameters at study entry ≥3000mm2, cardiac comorbidity, bone marrow infiltration, and atypical or leukemic cells in peripheral blood at study entry. The simplified CRS-RS.5p concentrates on the first five parameters utilizing the same weighting scheme as in CRS-RS. The primary objective of the GO43921 study is to determine if a five-baseline-parameter model can predict Grade ≥2 CRS events after the first dose of glofitamab. Seven studies were identified in which pts with aggressive NHL (first line and R/R, excluding mantle cell lymphoma) were treated with glofitamab step-up dosing as either monotherapy or part of combination therapy. In most studies, pts are pretreated with 1000mg obinutuzumab, with intravenous glofitamab being administered on Days (D) 1 (2.5mg) and 8 (10mg) of C1, then at the target dose (30mg) from C2D1 onwards. Several combination studies started glofitamab step-up dosing after multiple cycles of R-CHOP (glofitamab D8 [2.5mg], D15 [10mg], target dose D8 [30mg] in the following cycle). Prospective data has been collected since December 2021, and retrospective data from included studies were analyzed to increase the sample of Grade ≥2 CRS events. Here, CRS-RS.5p is compared with CRS-RS for the NP30179 dataset, and CRS-RS.5p performance in the ongoing GO43921 study is analyzed. Results: In NP30179, data acquired since the introduction of CRS-RS (n=44) showed that CRS-RS and CRS-RS.5p identified as high risk the same pts who experienced Grade ≥2 events (Figure A; four Grade 2, one Grade 3). In the whole NP30179 validation cohort, both CRS-RS and CRS-RS.5p achieved negative predictive values of 0.97 (n=148, Standard error=0.02). For GO43921, at the clinical cut-off date (June 6, 2022), the CRS-RS.5p model had predicted all 11 of the Grade ≥2 CRS events in the second-line or later setting for pts with aggressive NHL (n=123, with data collected retrospectively for 82 pts; Figure B). The association of the CRS-RS.5p score with the ASTCT CRS Grade after the first dose of glofitamab was similar across all available data sets from NP30179 and GO43921 (logistic regression; p<0.001 for NP30179, p=0.004 for GO43921). No influence of steroid premedication class on CRS-RS performance was observed. A low-risk group (CRS-RS.5p <4.0) comprised 48% of pts in NP30179 and 46% in GO43921. Conclusions: Data from the CRS-RS.5p model showed identical performance with the previous 8-parameter CRS-RS model in predicting which pts with aggressive NHL are most at risk of Grade ≥2 CRS after the first dose of glofitamab. With reduced complexity, the CRS-RS.5p has a potential application as a practical, clinical decision tool to guide hospitalization for pts at high risk of Grade ≥2 CRS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of an Off-the-Shelf Chimeric Antigen Receptor (CAR)-T Cell Therapy for T-Cell Acute Lymphoblastic Leukemia (T-ALL) without Gene Editing

CAR-T cell therapy has revolutionized the treatment of B-lineage ALL and other B-cell malignancies, but the development of cell therapies for T-cell malignancies has lagged behind. For T-ALL, CD7 is an excellent CAR target because it is consistently and highly expressed in leukemic cells and is retained during chemotherapy and at relapse. However, CD7 is also expressed in normal T-cells and the expression of anti-CD7 CARs triggers T-cell fratricide, resulting in a marked decrease in viable and/or effective CAR-T cells. We developed an anti-CD7 CAR-T cell product (PCART7) by utilizing a CD7 protein expression blocker (PEBL) to downregulate CD7 surface expression and prevent fratricide. PEBLs consist of a target binding domain fused to an intracellular retention domain that enables relocalization of surface proteins intracellularly (Png et al. Blood Adv 2017; Kamiya et al. Blood Adv 2018; Kamiya et al., JCI 2019). Autologous PCART7 cells lacking surface CD7 and expressing anti-CD7 CARs can be effectively produced under cGMP conditions and the technology is being tested in clinical trials for patients with relapsed/refractory T-ALL or T-cell lymphoblastic lymphoma. Generation of autologous CAR-T cells, however, may be hampered by the presence of a high percentage of leukemic cells in peripheral blood and/or low T cell numbers. An allogeneic anti-CD7 CAR-T cell product available "off-the shelf" would represent a useful tool to reduce leukemic burden and provide a bridge to hematopoietic cell transplant or to autologous CAR-T cell therapy. Because allogeneic T-cells can be activated upon recognition of host tissue antigens by their T-cell receptor (TCR)/CD3 complex, their infusion carries a risk of causing graft-versus-host disease (GvHD). We optimized a CD3 PEBL that can effectively downregulate surface TCR/CD3. We then developed a lentiviral-based process that allows simultaneous expression of CD3 and CD7 PEBLs together with anti-CD7 CAR. The PEBLs retained surface CD7 and TCR/CD3 intracellularly, thereby minimizing fratricide and abrogating TCR-mediated antigen recognition. PCART7 cells lacking TCR/CD3 were manufactured from healthy donor T-cells by using a double transduction with a bicistronic lentiviral vector delivering CD7 PEBL and anti-CD7 CAR (PCART7 LVV), and a second lentiviral vector delivering CD3 PEBL. Robust expansion of PCART7-CD3 PEBL cells was observed and more than 90% of the cells were of the desired CAR+CD7-CD3- phenotype. Following depletion of residual CD3/TCRαβ+ cells, CD3- cell purity was >99% (Fig. 1A). These results were reproduced across multiple donors. The efficient downregulation of surface TCR/CD3 by CD3 PEBL was similar to that achieved by CRISPR knockout of the TCR alpha chain locus (TRAC) and completely abrogated activation in response to TCR stimulation. In vitro short- and long-term assays confirmed strong cytotoxic potency against CD7+ leukemic cells but not CD7- target cells. Importantly, PCART7 cells were equally cytotoxic regardless of whether they were co-transduced with CD3 PEBL. In a xenograft model using the CCRF-CEM T-ALL cell line, PCART7-CD3 PEBL cells, like PCART7 cells, effectively suppressed tumor growth and improved survival (Fig. 1B). This study demonstrates the feasibility of using two PEBLs simultaneously for intracellular protein retention without perturbing critical CAR-T cell functions. The use of PEBL technology to generate allogeneic anti-CD7 CAR-T cells fits seamlessly in cGMP manufacturing protocols and avoids the risk of genotoxicity arising from the use of gene-editing techniques. Because the resulting allogeneic anti-CD7 CAR-T cells had high potency and were devoid of alloreactivity potential, they represent an attractive new tool for off-the-shelf CAR-T cell therapy of T-ALL and other CD7+ malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histone Deacetylase Inhibitor I3 Induces the Differentiation of Acute Myeloid Leukemia Cells with t (8; 21) or MLL Gene Translocation and Leukemic Stem-Like Cells.

Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by the clonal expansion and differentiation arrest of leukemic cells in peripheral blood and bone marrow. Though the treatment using cytarabine-based protocol for AML patients with t (8; 21) translocation has improved the 5-year overall survival rate, drug resistance continues to be the principal limiting factor for the cure of the disease. In addition, very few AML patients with mixed lineage leukemia gene rearrangements (MLLr) have a desirable outcome. This study evaluated the cell differentiation effect of a potent HDAC (histone deacetylase) inhibitor, I3, and its possible mechanism on the AML cells with t (8; 21) translocation or MLLr and leukemic stem-like cells (Kasumi-1, KG-1, MOLM-13, and THP-1). I3 exhibited efficient anti-proliferative activity on these cells via promoting cell differentiation, accompanied by the cell cycle exit at G0/G1. Importantly, I3 showed the properties of HDAC inhibition, as assessed by the acetylation of histones H3 and H4, which resulted in blocking the activation of the VEGF (vascular endothelial growth factor)-MAPK (mitogen-activated protein kinase) signaling pathway in the Kasumi-1 cell line. These data demonstrate that I3 could be a potent chromatin-remodeling agent to surmount the differentiation block in AML patients, including those with t (8; 21) translocation or MLLr, and could be a potent and selective agent for AML treatment.

Autologous CD7-targeted CAR T-cell therapy for refractory or relapsed T-cell acute lymphoblastic leukemia/lymphoma.

7035 Background: Refractory or relapsed (r/r) T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) has a poor prognosis. Our previous report showed that donor-derived CD7 CAR-T cell therapy could induce remission of r/r T-ALL, but graft-versus-host disease (GVHD) occurred in some patients (Pan et al. J Clin Oncol 2021;39:3340-3351). We recently conducted a phase I trial to evaluate autologous CD7 CAR-T therapy in r/r T-ALL/LBL, which may avoid GVHD while showing the efficacy; here we report the early result with the approval of the Data and Safety Monitoring Committee and Institutional Review Board (IRB). Methods: The patients who had CD7+ r/r T-ALL/LBL and no leukemia cells in peripheral blood were eligible for this phase I trial (NCT04840875). The CD7 CAR construct included an endoplasmic reticulum anchor domain fused to a CD7 binding domain to prevent CD7 expression on cell surface, which contributed to minimize CAR T cell fratricide. CAR T product was checked to ensure lack of tumor contamination before infusion. The trial followed a “3+3” dose escalation process. Adverse events and efficacy were primary and secondary endpoints. Results: A total of 5 patients (Pt1-5), with a median age of 3.8 years (range, 1.9-13), were enrolled between Sept 2021 and Jan 2022. At enrollment, Pt1 had mediastinal mass and blasts in pleural fluid; Pt4 was in central nervous system (CNS)-3 status; Pt2, 3 and 5 had marrow disease with a median burden of 1.35% (range, 0.07%-7.31%). Pt1, 2 and 3 received 5×105 (±20%) cells/kg; Pt5 received 1×106 (±20%) cells/kg; Pt4 received cells below the target dose. Three patients had cytokine release syndrome (CRS), including one grade 3; the median onset was on day 5 (range, 1-9) with the median duration of 4 days (range, 3-14). No patient had neurotoxicity, GVHD or infection. Grade 3-4 hematological toxicity occurred in all five patients, which recovered to grade 2 within 30 days. On one month post-infusion, four patients achieved complete remission, and one patient (Pt4) still had leukemia cells in the cerebrospinal fluid. With the median follow-up time of 62 days (range, 35-136), one patient (Pt1) underwent stem cell transplantation (SCT) on 2.9 months post-infusion, and had a CD7- relapse on 1.4 months post-SCT; the other three responders remained in MRD- CR. In the four patients who received target dose, the median peak CAR T cell count in peripheral blood was 4.27×102/uL (range, 2.49-5.61) by flow cytometry, and all five patients had detectable CAR transgene by PCR on their last visits. There was a decrease of CD7-positive normal T cells and an increase of CD7-negative counterparts in all responders. Conclusions: Autologous CD7 CAR T cell therapy was safe and effective in the induction of remission in r/r T-ALL/LBL patients, without signs of GVHD. Longer follow-up time of more cases will be used to further evaluate this therapy. Clinical trial information: NCT04840875.

Impact of anti-diabetic sodium-glucose cotransporter 2 inhibitors on tumor growth of intractable hematological malignancy in humans

Under the dysfunction of mitochondria, cancer cells preferentially utilize both glycolytic and pentose phosphate pathways rather than electron transport chains to desperately generate adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), classically recognized as the Warburg effect. Based on this background, the present study tested the hypothesis that anti-diabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors would exert a tumor-suppressive impact on intractable human hematological malignancies via the modulation of glucose metabolism within cells and cell cycles. The level of mRNA for SGLT2 was remarkably elevated in leukemic cells from patients with adult T-cell leukemia (ATL), one of the most intractable blood cancers in humans, and as well as in two kinds of ATL cell lines (MT-1 and MT-2). Two kinds of SGLT2 inhibitors, Luseogliflozin and Tofogliflozin substantially suppressed the proliferation of MT-1 and MT-2 cells in both adherent and anchorage-independent culture conditions. Such a suppressive effect on tumor cell growth was reproduced by Luseogliflozin in leukemic cells in peripheral blood from patients with ATL. In MT-2 cells, both of SGLT2 inhibitors considerably attenuated glucose uptake, intracellular ATP levels, and NADPH production, resultantly enhancing cell cycle arrest at the G0/G1 phase. From the standpoint of metabolic oncology, the present study suggests that SGLT2 inhibitors would be a promising adjunctive option for the treatment of the most intractable human hematological malignancies like ATL.

Synergistic Activity of IDH1 Inhibitor Bay-1436032 with Azacitidine in IDH1 Mutant Acute Myeloid Leukemia

Background: Only about one third of AML patients with mutant IDH1 respond to IDH1 inhibitor monotherapy with a median response duration of 7 months, indicating the strong need for combination treatments. We have previously shown that BAY-1436032 is a highly effective oral pan-mutant IDH1 inhibitor, which has strong anti-leukemic activity in patient derived xenograft (PDX) models of IDH1 mutant leukemia in vivo. In the present study, we assessed the combination of azacitidine with IDH1 inhibitor BAY-1436032 in a preclinical PDX model of IDH1 mutated AML.Methods: We tested the activity of BAY-1436032 alone or in combination with escalating doses of azacitidine in IDH1 mutated and IDH1 wildtype AML patient cells in vitro evaluating the effects on colony formation, apoptosis, cell cycle, differentiation and global gene expression changes. Leukemic cells from an AML patient with mutated IDH1, NPM1, FLT3-TKD and NRAS were xenografted in immunocompromised mice and treatment was started 28 days after transplantation. The control groups were treated with either vehicle, BAY-1436032 150 mg/kg once daily p.o. continuously, or azacitidine 1 mg/kg once daily s.c. days 1-5, repeated once after 28 days. The test groups were treated with BAY-1436032 and azacitidine in the doses mentioned above either starting both drugs on day 1 (parallel group) or starting azacitidine on day 1 but BAY-1436032 on day 6 (sequential group). The treatment was stopped after 84 days.Results: Our IDH1 inhibitor reduced colony formation specifically in human IDH1 mutated AML cells, while IDH1 wildtype cells were not affected (IC50 100 nM). Combination of BAY-1436032 (100 nM) with azacitidine 100 nM further reduced colony formation by 50% in IDH1 mutated AML cells, while the IC50 for IDH1 wildtype AML cells was not reached even at 1000 nM azacitidine in the presence of 100 nM BAY-1436032. For in vivo experiments human AML cells from an IDH1 mutated AML patient were transplanted into sublethally irradiated NSG mice and treatment was started four weeks after transplantation. Leukemic cells in peripheral blood constantly increased in vehicle and azacitidine treated mice, while leukemic cells declined from week 4 until the end of treatment at week 12 in mice treated with BAY-1436032 alone as well as in the parallel and sequential combination groups treated with BAY-1436032 and azacitidine. However, in week 20 (8 weeks after stopping the BAY-1436032 treatment) leukemia relapsed in the BAY-1436032 alone as well as the sequential combination groups. Interestingly, the frequency of leukemic cells remained low in peripheral blood in mice treated in parallel with the combination of BAY-1436032 and azacitidine even at 24 weeks after starting the treatment (Figure 1). To obtain mechanistic insights into the efficacy of the combination treatment we treated human IDH1 mutant and IDH1 wildtype AML cells in vitro with either 100 nm BAY-1436032 or 100 nm azacitidine alone or in combination (in parallel). Vehicle treatment served as control. There were no significant differences in percentage of apoptotic cells between BAY-1436032 or azacitidine treatment as single agents or in combination. However, the proportion of cells in S phase of the cell cycle was synergistically decreased by the combination treatment compared with either monotherapy or vehicle. Gene expression profiling showed that the target genes of EGR1-3 and GFI1 transcription factors were upregulated by the combination treatment over BAY-1436032 treatment alone, while target genes of NFkB and its targets ELF1 and STAT1/3 were down regulated, suggesting synergy in induction of differentiation and inhibition of self-renewal of the combination treatment through distinct pathways.Conclusion: Our study provides the first evidence of synergistic activity of an IDH1 inhibitor with hypomethylating agents through inhibition of the cell cycle by dysregulating EGR, GFI1 and NFkB signaling, and strongly argues for simultaneous application of the IDH1 inhibitor BAY-1436032 with azacitidine in future clinical trials. Clinical development is ongoing with phase 1 studies using BAY-1436032 in IDH1 mutant solid tumors and AML.Figure 1: Development of human IDH1 mutated AML in peripheral blood of NSG mice treated with BAY-1436032 and azacitidine alone or in combination (PRL, in parallel; SEQ, sequential, i.e. first azacitidine days 1-5, then BAY-1436032 from day 6 onwards). [Display omitted] DisclosuresChaturvedi:Bayer Pharma AG, Berlin, Germany: Research Funding. Kaulfuss:Bayer Pharma AG, Berlin, Germany: Employment. Panknin:Bayer Pharma AG, Berlin, Germany: Employment. Wagner:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership. Jeffers:Bayer Pharma AG, Whippany, NJ, USA: Employment. Haegebarth:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership.

Genetic and Transcriptomic Characterization of Marrow Progenitor Cells in Patients with Chronic Lymphocytic Leukemia

Tumor formation is the result of a stepwise process through which cells acquire genetic alterations that may confer growth advantages to enable clonal expansion. One model in leukemia is that mutations accumulate in normal hematopoietic stem cells (HSC) leading to pre-leukemic cells which carry some, but not all, leukemia-specific mutations. Chronic lymphocytic leukemia (CLL) has been traditionally viewed as a mature B cell malignancy, however, recent studies have identified leukemia-specific mutations in CD34+ cells from either peripheral blood or bone marrow. These data raise the question of whether marrow progenitor cells in CLL are dysregulated even before commitment to the B cell lineage and, if so, what are the characteristics of such cells in CLL.To this end, we first quantified the absolute number of CD34+ cells from marrow collected from CLL patients (n=9) vs healthy adult volunteers (n=8). Using a florescence bead-based absolute quantification assay (BD Trucount), we detected a significant reduction of CD34+ progenitor cells in CLL compared to normal donors (Average: 55.6 vs 391/ml, p=0.002, Welch's t -test). To determine if this quantitative change was accompanied by changes in the cellular state of these cells, we performed low-input RNA sequencing (RNA-seq) from 7 marrow progenitor subpopulations derived from 5 CLL patients and 4 healthy donors, that were isolated on the basis of flow cytometric sorting. These subpopulations included HSC (Lin-CD34+CD38-CD90+CD45RA-), MPP (Lin-CD34+CD38+CD90-CD45RA-), MLP (Lin-CD34+CD38-CD90-CD45RA+), CMP (Lin-CD34+CD38+CD90-CD135+CD45RA-), MEP (Lin-CD34+CD38+CD90-CD135-CD45RA+), GMP (Lin-CD34+CD38+CD90-CD135+/-CD45RA+), and CLP (Lin-CD34+CD38+CD10+CD45RA+). This systematic analysis revealed both a CLL-specific transcriptome profile as well as unique expression signatures for each progenitor subpopulation. In total, 119 genes were identified as CLL progenitor cell-specific and were highly enriched for the regulation of mRNA catabolic processes and protein translation.To determine if leukemia-specific mutations are present in these progenitor cells and may contribute to dysregulation, we performed targeted deep-sequencing with low-input DNA derived from these subpopulations. We focused on marrow cells collected from 3 CLL patients, whose matched peripheral blood leukemia cells had previously undergone whole-exome sequencing (WES). Of the 30 somatic mutations identified by WES in CLL cells from Patient 1, we detected presence of 4 and 26 mutations in HSC and Pro-B/Pre-B cells, respectively. Likewise, we detected 4 of 34 CLL-specific mutations in HSC cells for Patient 2. In contrast, none of 10 mutations detected in Patient 3's CLL cells by WES were evident in the HSC subpopulation, but 9 of 10 mutations could be identified at the Pro-B/Pre-B stage. None of the 74 mutations from CLL cells from Patients 1-3 were detected in DNA extracted from marrow cells collected from 3 normal healthy donors.To confirm the presence of these somatic mutations in CLL progenitor cells, we utilized a microfluidics-based approach to perform multiplexed simultaneous targeted detection of up to 48 mutations and expression of up to 96 genes from cDNA of individual cells. We constructed panels of 96 genes based on the aforementioned RNA-seq data to confirm both identity of cellular differentiation stage and cellular source (normal vs CLL). We thus tested whether the single-cell marrow progenitor cells from CLL patients harbored leukemia-specific mutation while expressing progenitor cell genes. With this method, we validated the expression of 2 mutations in bona fide HSC at the single-cell level in Patient 1. Further validation studies of somatic mutation detection using low-input DNA and single-cell approach are ongoing.In summary, we present evidence of pre-leukemic cells at the HSC stage and these cells have increasing similarity to the cognate CLL at the pro/preB stage. Our DNA and single-cell analysis highlight the existence of CLL progenitor cells harboring leukemia-specific mutations while transcriptome profiling identified a CLL-specific gene signature associated with these pre-leukemic cells. We anticipate that further systematic analysis linking mutation, epigenetic changes, and dysregulated gene expression will enhance our understanding of the stepwise transformation and pathogenesis of this disease. DisclosuresNeuberg:Synta Pharmaceuticals: Other: Stock shares. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding. Brown:Janssen: Consultancy; Janssen Oncology: Honoraria; Redx: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Consultancy; Infinity Pharmaceuticals: Consultancy; Roche/Genentech: Consultancy; Astellas Pharma: Consultancy; Pfizer: Consultancy; Sun BioPharma: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria; Pharmacyclics: Consultancy; AstraZeneca: Consultancy. Wu:Neon Therapeutics: Consultancy.

Expression of YY1 in pro-B and T phenotypes correlation with poor survival in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, constituting 80% of all acute leukemias in minors. Despite the increase in the success of therapies, disease-free survival is over 80% in most cases. For the remaining 20% of patients, new strategies are needed to allow us to know and select those at greatest risk of relapse. We evaluated by immunohistochemistry the expression of the transcription factor YY1 and found that it is overexpressed in peripheral blood leukemia cells of pediatric patients with ALL with Pro-B and T phenotype compared to control samples. Over expression of YY1 was associated with a significantly lower chance of survival. We also evaluated by RT-PCR in bone marrow samples from ALL pediatric patients the association of YY1 expression with the percentage of blasts. High levels of YY1 were present in samples with higher percent of blasts in these patients. In addition, ALL pediatric patients with a poor response to therapy had higher levels at the nuclear level of YY1 than those who responded well to chemotherapy. In conclusion, our data suggest that YY1 could serve in pediatric ALL as markers of evolution and response for this disease, mainly in patients with pro-B and T immunophenotype. It is also suggested that YY1 is implicated in the expanse of blast in these patients.

Monoclonal Antibodies to Myeloid Differentiation Antigens: In Vivo Studies of Three Patients With Acute Myelogenous Leukemia

Three patients with acute myelogenous leukemia (AML) in relapse were treated with intravenous infusions of one or more purified murine monoclonal antibodies (MoAbs) specific for differentiation antigens on normal and malignant myeloid cells. Three of the MoAbs used were IgM immunoglobulins that react with glycolipids, while the fourth, an IgG2b, reacts with a protein antigen. Peripheral blood leukemia cell counts decreased significantly, but transiently, during treatment. Evidence of in vivo binding of each MoAb to leukemia cells was obtained, although two of the four MoAbs could not be detected in the plasma following infusion, perhaps due to circulating blocking factors. Antigenic modulation was not encountered in these studies. However, the induction of human antibody to murine MoAb was observed in one patient who was treated over a 70-day period. Toxicities encountered were minimal and included fever (3 patients), back pain (1 patient), and arthralgias and myalgias (1 patient). This is the first reported clinical trial of (1) IgM MoAbs, (2) MoAb therapy in patients with AML, (3) combinations of MoAbs directed toward different myeloid differentiation antigens, and (4) MoAbs directed to glycolipids. The relative lack of toxicity and the positive effects of MoAb treatment in the reduction of leukemia cell counts permit the continued study of more innovative approaches to the treatment of AML with MoAbs.

Overcoming NOTCH1-Driven Chemoresistance in T-Cell Acute Lymphoblastic Leukemia Via Metabolic Intervention with Oxphos Inhibitor

Overcoming NOTCH1-Driven Chemoresistance in T-Cell Acute Lymphoblastic Leukemia Via Metabolic Intervention with Oxphos Inhibitor

PHF6 Restricts AML Acceleration By Promoting Myeloid Differentiation Genes in Leukemic Cells

Acute myeloid leukemia is caused by the accumulation of mutations in hematopoietic stem and myeloid progenitor cells, resulting in increased self-renewal, inhibition of differentiation, and aberrant proliferation. Although genomic studies have comprehensively identified genes that are mutated in acute leukemias, the functional roles of many of them, and the consequences of their mutations, remain poorly understood. PHF6 (PHD-finger protein 6) is an X-linked gene that is mutated in 3.2% of de novo AML, 4.7% CMML, 3% MDS, and 1.6% CML patients. Two-thirds of somatic mutations in PHF6 are frameshift and nonsense mutations distributed throughout the gene body, resulting in loss of PHF6 protein. One-third of the mutations are point mutations clustered in the ePHD2 (extended PHD) domain, and the consequence of these mutations on PHF6 function is unknown. The functional role of PHF6 and the mechanism by which PHF6 mutations accelerate AML has not yet been determined. In this study, we delineate the cellular and molecular function of PHF6 in AML using in vitro and in vivo models. In agreement with recently published reports, we found that pan-hematopoietic deletion of Phf6 using the Vav-Cre recombinase system gave competitive transplantation advantage to HSCs, with sustained multi-lineage reconstitution without exhaustion over three rounds of serial transplantations, demonstrating that Phf6 represses HSC self-renewal. However, loss of Phf6 alone was insufficient to cause hematopoietic malignancy in the mouse model when monitored for one year. To determine the function of PHF6 in AML progression, we transduced cKO (Vav-Cre; Phf6 flox) or WT (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HOXA9 (WT+HOXA9 and cKO+HOXA9), and transplanted into irradiated recipient mice. The resulting HOXA9-driven AML was greatly accelerated in the Phf6 cKO background, with recipient mice succumbing faster (median survival 119 days) as compared to recipients transduced with HOXA9-transduced WT cells (median survival &amp;gt;180 days, p=0.003) (Fig 1A). This was also reflected by an increase in the number of circulating immature leukemic cells in peripheral blood at earlier timepoints. HOXA9-transduced cKO cells showed higher serial replating ability in an in vitro colony forming assay as compared with HOXA9-transduced WT cells (Fig 1B). We further investigated the molecular function of PHF6 using the THP-1 human AML cell line. PHF6 is a chromatin-binding protein with two ePHD domains, and its binding partners and pattern of chromatin occupancy are unclear. Using ChIP-Seq, we identified that PHF6 occupies enhancers, and its peaks show striking alignment with the peaks of the key myeloid transcription factors (TFs) RUNX1, PU.1, and IRF8 (Fig 1C). To assess the effect of the clinically relevant point mutation R274Q (in the ePHD2 domain) on the transcriptional effects produced by PHF6, we first generated a PHF6 KO clone from the THP-1 cell line, and then re-expressed either WT PHF6 or R274Q-mutant PHF6 in this KO line. Re-expression of WT PHF6 rescued the extensive gene expression changes produced by its knockout, but R274Q-mutant PHF6 was unable to produce any gene expression changes, indicating that it is a "transcriptionally dead" mutant (Fig 1D). Gene Ontology analysis of transcriptome changes induced by WT PHF6 showed that PHF6 promotes the expression of myeloid differentiation gene sets. In summary, PHF6 restricts AML progression by binding enhancers with key myeloid TFs, and promoting the expression of myeloid differentiation genes. R274Q mutation renders PHF6 unable to exert any downstream expression changes, indicating that the ePHD2 domain (where R274 is located, clustered with other amino acids showing point mutations in hematopoietic malignancies) is critical for PHF6 function, and likely mediates important functional interactions with chromatin partners. Our future work will involve dissection of the sequence of molecular events governed by PHF6 following enhancer occupancy, and the role of PHF6 in repressing AML self-renewal and promoting differentiation. Disclosures No relevant conflicts of interest to declare.