Burden In Bone Marrow Research Articles

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Dual Targeting Novel WDR5/ATAD2 Oncogenic Signaling through CK2/Ikaros Axis Demonstrates Synergistic Efficacy in T-ALL

Background WD repeat-containing protein 5 (WDR5) is highly expressed in human cancers and its high expression is linked with adverse prognoses. However, the oncogenic roles of WDR5 and underlying mechanisms are undefined in T-cell acute lymphoblastic leukemia (T-ALL). Casein Kinase II (CK2) is a pro-oncogene, and CK2-mediated IKAROS dysfunction plays critical roles in high-risk ALL, and CK2 inhibitor CX-4945 has the anti-leukemic effect by restoring IKAROS function. This study aims to explore the synergistic anti-leukemic efficacy of dual targeting novel WDR5/ATAD2 oncogenic signaling via the CK2/IKAROS axis in T-ALL. Methods Clinical samples from 38 T-ALL patients and 32 healthy controls were obtained from Zhongda Hospital Southeast University. The expression of WDR5 and ATAD2 were tested by q-PCR and Western blot. RNA-seq was performed after CEM cells were treated with WDR5 inhibitor (OICR-9429), CX-4945 and vehicle control for 72 hours. The human leukemia xenograft mouse model is established by intravenously injected with WDR5 knockdown CEM (CEM-shWDR5) cells and shRNA control (CEM-shNC) into NSG mice. Mice were treated for 25 days in 4 groups [CEM-shNC + vehicle, CEM-sh WDR5 + vehicle; CEM-shNC + CX-4945 (100 mg/kg via gavage), CEM-sh WDR5 + CX-4945]. Leukemia burden in mice spleen and bone marrow was analyzed by flow cytometry. The survival of mice was compared by the Kaplan-Meier method. The synergistic effect of CX-4945 and OICR-9429 was analyzed by CCK8 assay. This study was approved by the Ethics Committee of Zhongda Hospital Southeast University Results WDR5 ishighly expressed in T-ALL patients compared with normal controls( P<0.001) and is higher in the relapsed samples than paired newly-diagnosed controls ( P<0.05) (Fig. 1A-B). WDR5 knockdown in T-ALL cells significantly suppressed cell proliferation and induced cell cycle arrest in vitro ( P<0.01) (Fig. 1C), and in vivo leukemia development (Fig. 1D). Also, WDR5 inhibitor OICR-9429 induces cell proliferation and cell cycle arrest in the cells. RNA-seq revealed that ATAD2 is downregulated upon OICR-9429 treatment and indicated that ATAD2 is the downstream target of WDR5. WDR5 knockdown suppresses ATAD2 expression ( P<0.01) (Fig. 1E). In addition, ATAD2 ishighly expressed in T-ALL patients versus normal controls ( P<0.05) (Fig. 1F), and ATAD2 silencing significantly suppresses cell proliferation and induces cell cycle arrest of T-ALL cells ( P<0.01) (Fig. 1G). These data indicate WDR5/ATAD2 signaling act as a novel potential oncogenic pathway in T-ALL. Furthermore, we found that IKAROS transcriptionally suppresses WDR5 expression, and CK2-mediated hyperphosphorylation of IKAROS is one of the major mechanisms of IKAROS dysfunction in ALL. CX-4945 strongly suppresses WDR5 expression in an IKAROS-dependent manner (Fig. 1H-J). The synergistic effect of CX-4945 with WDR5 disruption on cell proliferation suppression and cell cycle arrest was observed in T-ALL both in in vitro and in vivo xenograft mouse model. The combination of CX-4945 with WDR5 disruption significantly prolonged the survival of mice compared with single CX-4945 [55(52-58) days vs. 38.5(36-41) days， P<0.001] and single WDR5 disruption control [55(52-58) days vs. 41.5(39-44) days, P<0.001] (Fig. 1K). Consistently, the combination significantly reduces the size and weight of the spleen (Fig. 1L), % human CD45+ T-ALL cells in the spleen (Fig. 1M) and bone marrow (data not shown), and the expression of WDR5 and ATAD2 in the spleen versus single treatment controls ( P<0.01) (Fig. 1N-P). Finally, a synergistic effect of CX-4945 with OICR-9429 on cell proliferation suppression and down-regulation of WDR5 and ATAD2 was observed in the primary leukemic cells from T-ALL patients (Fig. 2A-D). Together, these data indicated the synergistic efficacy of CX-4945 with OICR-9429 on T-ALL and the novel model was summarized in Fig.2E. Conclusion In this study, we identified a novel WDR5/ATAD2 oncogenic signaling regulated by CK2/IKAROS axis in T-ALL. Our study reveals a model that dual targeting WDR5/ATAD2 signaling through direct inhibiting oncoproteins and via CK2/IKAROS axis to transcriptionally repress the oncoprotein to achieve synergistic efficacy. Our results further highlight the combination of CX-4945 with WDR5 inhibition is a potential option for the therapy of T-ALL patients.

Debio 1562M, a Next Generation Antibody Drug Conjugate (ADC) Targeting CD37 for AML and MDS Treatment

CD37 is a trans-membrane protein specifically expressed on normal hematopoietic tissues such as B cells, neutrophils and macrophages, and has demonstrated roles in adhesion, cell migration (de Winde et al, 2018; Wee et al, 2015), survival and apoptotic signals (Lapalombella et al, 2012). Increased expression of CD37 has been observed in various hematological cancers such as lymphomas, Chronic Lymphocytic Leukemia (CLL) and Acute Myeloid Leukemia (AML) (Beckwith et al, 2014; Deckert et al, 2013; Pereira et al, 2015; Yan et al, 2021). Higher expression of CD37 was also associated with poor patient outcome in AML (Zhang et al, 2020; Larkin et al, ASH 2018). Debio 1562M is a second-generation novel ADC directed against CD37 and including Debiopharm's proprietary Multilink cleavable linker-payload technology. Multilink improves manufacturing to achieve homogenous high drug antibody ratio while retaining excellent solubility as well as favorable biologic characteristics with preserved internalization kinetics on target cells. Debio 1562M is conjugated to 8 molecules of a tubulin-binding agent and despite cleavable linker that ensure effective intracellular delivery of toxic payload, we show sustained plasma stability in vitro and in vivo with no premature extracellular release. In this work, we report Debio 1562M specificity and potent antitumor activity in multiple AML models both in vitro and in vivo. First, in our in vitro cytotoxicity assay, Debio 1562M displayed nano- to picomolar anti-proliferative activity in both AML cell lines and primary samples. Out of 30 primary samples tested, 20 were sensitive to Debio 1562M with IC50 ranging from 1 to 10nM, this regardless of their mutation status, subtype or medical history. Interestingly, we observed that AML cells have high internalization capability and therefore relatively low levels of surface CD37 is sufficient for cytotoxic activity. The specificity of Debio 1562M was demonstrated by using 3 different CD37 knock out AML cell lines which were generated by CRISPR-Cas9 technology. Parental and CD37 KO cells were treated for 72h with a dose range of Debio 1562M and viability was measured (Figure 1). While Debio 1562M has an IC50 below 1nM in all 3 parental cell lines, its activity is reduced by more than 2 log in the KO cell lines. In vivo, in 3 different AML cell line xenograft mouse models, a single injection of Debio 1562M at 5mg/kg induced complete tumor regression and all mice remained alive at study termination, more than 60 days after treatment, except one mouse in one model. In a patient derived xenograft (PDX) model, the same dose of Debio 1562M significantly reduced tumor burden in blood and bone marrow with sustained activity 42 days after treatment (37% and 98% of blasts in blood and bone marrow respectively in control animals versus 0% and 3% in treated mice, pvalue<0.001). Furthermore, we evaluated the activity of Debio 1562M over venetoclax and azacytidine combination in the MV4-11 Luciferase xenografted mouse model. NCG mice were inoculated with 1x10 7 cells in the tail vein and randomization in groups of 8 mice was performed 14 days later. Mice received either one administration of Debio 1562M at 5mg/kg or a combination of venetoclax (QDx14 100mg/kg) and azacitidine (QDx5 3,5mg/kg). Complete regression of the tumor was observed in all mice after Debio 1562M administration and all mice survived at study termination, 75 days after treatment while none survived after 56 days when treated with venetoclax and azacitidine combination (Figure 2). The toxicology profile of Debio 1562M in mice showed expected clinical signs related to the payload's off target toxicity but with a significant safety margin. Interestingly, no peripheral neuropathy symptoms were detected, as commonly observed for this class of payload. Additionally, Multilink TM-payload construct alone was not associated with any signs of toxicity. On-target toxicity profile couldn't be evaluated as Debio 1562M isn't cross reactive with mouse CD37, however 1 st generation CD37 ADC has already demonstrated safety and tolerability in Phase 1 and 2 clinical trials.Overall, our data confirm the relevance of CD37 targeting for AML treatment. The excellent antitumor activity and tolerability of Debio 1562M in several pre-clinical models now pave the way for upcoming clinical development.

Identifying a core protein signature of small extracellular vesicles derived from B-cell precursor acute lymphoblastic leukaemia.

Acute paediatric leukaemia is diagnosed and monitored via bone marrow aspirate assessment of blasts as a measure of minimal residual disease. Liquid biopsies in the form of blood samples could greatly reduce the need for invasive bone marrow aspirations, but there are currently no blood markers that match the sensitivity of bone marrow diagnostics. Circulating extracellular vesicles (EVs) represent candidate biomarkers that may reflect the blast burden in bone marrow, and several studies have reported on the utility of EVs as biomarkers for adult haematological malignancies. Increased levels of EVs have been reported for several haematological malignancies, and we similarly report here elevated EV concentrations in plasma from paediatric BCP-ALL patients. Plasma EVs are very heterogeneous in terms of their cellular origin, thus identifying a cancer selective EV-marker is challenging. Here, we undertook a reductionistic approach to identify protein markers selectively associated to plasma EVs derived from BCP-ALL patients. The EV proteome of primary BCP-ALL cell-derived EVs were compared against EVs from healthy donor B cells and the BCP-ALL cell line REH, and further against EVs isolated from plasma of healthy paediatric donors and paediatric BCP-ALL patients. With this approach, we identified a signature of 6 proteins (CD317, CD38, IGF2BP1, PCNA, CSDE1, and GPR116) that were specifically identified in BCP-ALL derived EVs only and not in healthy control EVs, and that could be exploited as diagnostic biomarkers.

Increased clonal hematopoiesis mutational burden and accelerated dynamic evolution of circulating clones in patients with ischemic heart failure

Abstract Background Clonal hematopoiesis (CH) - defined as the age-related acquisition of mutations in hematopoietic stem cells giving rise to mutated circulating leukocytes - is associated with increased risk of death in patients with heart failure with reduced ejection fraction (HFrEF). While the mutational landscape of CH in peripheral blood (PB) of patients with HFrEF is well characterized, the mutational burden in bone marrow (BM) samples of patients with HFrEF as well as its dynamics over time have not been investigated. Methods/Results We analyzed matched BM and PB cells from 68 patients with ischemic HFrEF by deep error-corrected targeted sequencing of 56 CH-driver genes with a detection threshold of VAF ≥0.5%. In PB, 56/68 patients carried at least one CH mutation, whereas 63/68 patients had mutations in BM. The absolute numbers of detectable mutations were significantly higher in BM compared to PB (215 mutations vs 120 mutations; p<0.0001) (Fig. 1A). Likewise, the cumulative VAF was significantly higher in BM compared to PB (3.83 +/- 0.5% vs 3.0% +/-0.7%; mean +/- SEM, p = 0.0025) (Fig. 1B). However, there was a strong correlation between mutation load in PB and BM (r = 0.54, p<0.0001). Among the two most common mutations, DNMT3A mutations were either present in BM only or both compartments (PB only/BM only/ both compartments 2/12/10), whereas TET2 mutations were strikingly enriched in BM cells (PB only/BM only/both compartments 0/16/2). To study the dynamic evolution of CH clones in patients with HFrEF, we serially analyzed PB samples from 19 patients. At baseline, 16/19 patients carried at least one mutation in a CH driver gene and a total of 31 mutations. After a median follow-up of 12 months, 19/19 patients had at least one detectable mutation and a total of 43 mutations were identified (mean number of mutations per patient 1.63 +/- 0.033 vs 2,26 +/- 0.27; mean +/- SEM, p = 0,0410), indicating an increased mutation load over time (Fig. 2A). Moreover, cumulative VAF increased from 1.59 +/-0.36 % to 2.01 +/- 0.38 %). Conclusion Patients with ischemic HFrEF display a high burden of CH mutations compared to values reported for healthy individuals, which is further amplified in the bone marrow compartment. Clones with new mutations arise rapidly in longitudinal samples of patients with HFrEF and clonal expansion occurs with high momentum. The overrepresentation of mutations in the BM is indicative for augmented DNA damage in the hematopoietic stem cells of patients with HFrEF, while the rapid expansion of mutated circulating clones over time suggests that heart failure itself may expedite the expansion of hematopoietic cells harboring CH-driver mutations.

LSD1 Inhibition Synergizes with Venetoclax in Acute Myeloid Leukemia By Targeting Cellular Metabolism

Introduction: Acute myeloid leukemia (AML) is a hematological neoplasm with poor clinical outcomes. The introduction of the BCL2 inhibitor venetoclax in combination with hypomethylating agent has improved response rates in older patients with AML by targeting the metabolism of leukemic stem cells (LSCs). Resistance, however, is unfortunately still common. Thus, there remains a need to enhance the clinical efficacy of venetoclax by combining it with novel agents. In diverse AML models, lysine-specific demethylase 1 (LSD1) inhibition promotes cellular differentiation and reduces LSCs. We therefore asked if LSD1 inhibition could enhance the efficacy of venetoclax in the treatment of AML. Methods: We have studied the effect of single-agent and co-treatment with venetoclax and LSD1 inhibitor bomedemstat on cell proliferation in human AML cell lines (N=3), Hoxa9/ Meis1(H9M)- and MN1-transformed murine myeloid progenitor lines as well as primary AML patient samples (N=5). We have analyzed the effect of co-treatment with these two compounds on functional cellular bioenergetics in MOLM-13 cells using Seahorse extracellular flux analyzer and followed up by performing NMR spectrometry to study changes in metabolite abundance. Further, we have studied the efficacy of this drug combination in vivo in patient-derived xenograft (PDX) AML models (N=2). Results: Human AML cell lines showed a differential cytocidal activity at 96 hours (h) to venetoclax with MOLM-13 (IC 50 0.04 µM) being most sensitive and OCI-AML3 most resistant (IC 50 9.81 µM). A similar range (between 0.15 µM and 7.3 µM) was also observed in the primary AML samples. For bomedemstat, we observed a time-dependent response where the IC 50 decreased over time, in particular, in the most sensitive model systems. In murine H9M cells, the IC 50 decreased from 30.43 µM at 48 h to 0.034 µM at 96 h and further down to 0.0068 µM at 168 h. Combining bomedemstat and venetoclax, the IC 50 for venetoclax decreased; and synergism was observed in MOLM-13 cells, the most sensitive AML model, but no synergism was observed in venetoclax-resistant cells. Synergism was also observed in MN1 cells as well as 3 of the 5 primary AML samples studied (Table 1). To investigate the mechanism of the observed synergism, we explored the effect of these treatments on metabolic pathways using the Seahorse extracellular flux analyzer. LSD1 inhibition had no effect on oxidative phosphorylation (measured as oxygen consumption rate (OCR)). However, glycolysis, measured as proton efflux rate (PER), was significantly reduced from 102.88 ± 11.03 pmol/min to 54.17 ± 6.18 pmol/min (P <0.01) after 96 h of treatment with 1μM bomedemstat. With the combined treatments, we observed a reduction in both OCR and PER. This synergistic effect was further investigated by studying metabolite abundance using NMR spectrometry where co-treatment showed a reduction in metabolite abundance across a broad spectrum of metabolic pathways (Fig. 1). To determine if the observed combined effect of bomedemstat and venetoclax could enhance activity against AML cells in vivo, we employed a PDX model with two primary AML samples. In the first model system (monosomy 7), co-treatment resulted in a significant reduction in human CD45-positive cells as compared to vehicle (P <0.001) and single-agent treatments with venetoclax (P= 0.011) and bomedemstat (P <0.001). Similarly, in the second PDX model ( NPM1-mutated, FLT3-ITD positive), the co-treatment exhibited a significant reduction in leukemic burden in bone marrow as compared to vehicle (P=0.003) and single-agent treatments with venetoclax (P= 0.024) and bomedemstat (P < 0.001) (Fig. 1). Conclusion: The combination of bomedemstat and venetoclax had synergistic cytocidal effects on AML cell line and primary AML cells in vitro. It significantly reduced the leukemic burden in PDX AML models and synergistically downregulated cellular energy metabolism. These findings suggest that combining venetoclax with LSD1 inhibition holds promise as a combination treatment in AML and warrants further clinical investigation.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Long-Term Follow-up of Humanized and Murine CD19 CAR-T Cell Therapy for B-Cell Malignancies

Aim: CD19 chimeric antigen receptor T cell (CAR-T) therapy is a revolutionary treatment for relapsed/refractory (R/R) B-cell hematologic malignancies. However, the safety and efficacy of murine CAR-T cells which have been widely used as commercialized products still need to be improved. Similarly, murine CAR-T encountered the problem that CAR-related immunogenicity existed after the infusion, which led to the failure of the re-infusion of CAR-T cells. As an improved generation of murine CAR-T cells, whether humanized CAR-T therapy can overcome the above shortcomings remains to be explored. Methods: In this study, the differences in safety, efficacy, and long-term follow-up results of murine and humanized CD19 CAR-T cells in patients with B-cell malignancies were collected and analyzed. A total of 130 patients were enrolled, including 35 patients with murine CAR-T and 95 with humanized CAR-T cells (NCT 02965092, NCT 04008251). Results： The proportion of patients with cytokine release syndrome (CRS) in the murine and humanized groups was 54.3%（19/35） and 61.1%（58/95）, respectively. A significantly higher proportion of patients suffered from severe CRS in the murine group than that in the humanized CAR-T group (17.14% vs. 7.4%, P=0.0233), and one patient in each group died of Grade 5 CRS. The incidence of grade 1-2 Immune effector cell-associated neurotoxicity syndrome (ICANS) was 11.4% (murine: 4/35) and 5.3% (humanized: 5/95), but high-grade ICANS was not observed. Among patients receiving murine CAR-T cells, 80% achieved objective response (OR), 71.43% achieved complete response (CR) and the CR rate of patients with leukemia was 74.19%. The OR rate and CR rate of patients in the humanized group were 75.79% and 63.16%, respectively, while the CR rate of patients with leukemia receiving humanized CAR-T cells was 89.58%. The median follow-up time of patients in the murine and humanized groups was 7 months (0.6-75 months) and 5.5 months (0.5-34 months), respectively. The median progression-free survival (PFS) of patients with murine CAR-T cells was 11 months and that of patients with humanized CAR-T products was 12 months. Both of the median overall survival (OS) were not reached. Among the 48 patients with a bone marrow burden over 20% at baseline, humanized CAR-T therapy was associated with a higher OS rate (84.64% vs. 59.26%, P=0.1479) and a significantly improved PFS (43.94% vs. 33.33%, P=0.0217, Figure 1). Bridging transplantation was an independent factor in prolonging OS (x 2=8.017, p=0.0046) and PFS (x 2=6.584, p=0.0103) of patients with leukemia. Neither species of chimeric antigen receptor nor common risk factors such as age, recurrence, bone marrow burden, BCR-ABL fusion gene had significant effect on patients' long-term follow-up outcomes (Table 1). Four patients received CAR-T therapies more than once. Three of them still achieved varying degrees of long-term remission after multiple humanized CAR-T infusions. However, one patient relapsed as soon as one month after his second infusion of murine CAR-T cells. Conclusion: Results indicate that humanized CAR-T therapy showed better safety and durable efficacy, especially in patients with higher tumor burden in bone marrow. Moreover, it could overcome immunogenicity-induced CAR-T resistance, providing treatment options for patients' failures of CAR-T therapies.

Combining Menin and MEK Inhibition to Target Poor Prognostic KMT2A-Rearranged RAS Pathway-Mutant Acute Leukemia

Approximately 10% of all acute leukemias harbor a chromosomal translocation involving the Mixed Lineage Leukemia 1 ( MLL1/ KMT2A) gene locus with over 90 fusion partners. These fusions are especially prevalent in pediatric acute leukemias. The resulting KMT2A fusion proteins (KMT2A-r) drive leukemogenic gene expression through an interaction with a chromatin complex that includes the scaffold protein menin, giving rise to aggressive acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In infant ALL, frequently characterized by KMT2A-r, the presence of RAS pathway mutations has been associated with a poor prognosis. The prognostic impact of RAS pathway mutations in pediatric KMT2A-r AML is less established. We thus analyzed data from 1605 pediatric patients with AML treated on the Children's Oncology Group (COG) AAML0531 and AAML1031 studies, with 336 of these having KMT2A-r. 51% (170/336) of these patients had mutations in NRAS, KRAS or PTPN11. The combination of KMT2A-r with RAS pathway mutations was associated with worse EFS and OS, HR=1.71 (p<0.001) for EFS and OS multivariable models. Given the prevalence of RAS pathway mutations co-occurring with KMTA-r, and the high-risk leukemia associated with this mutational profile, we sought to identify efficacious targeted drug combinations for this subset of childhood leukemia. Early clinical trial data for the menin inhibitor revumenib (SNDX-5613) demonstrated efficacy in KMT2A-r leukemias. Responding patients, however, typically develop progressive disease with a subset having mutations in MEN1 the gene encoding menin. Effective combination treatments for full efficacy of this targeted therapy are needed. We evaluated the combination of RAS/MAPK targeting using the MEK1/2 inhibitor selumetinib with VTP-50469, a close analog of revumenib, in pediatric leukemias harboring both KMT2A-r and RAS mutations. Treatment of AML and ALL cell lines and cells from an AML patient-derived xenograft (PDX) model in vitro with this drug combination resulted in a synergistic decrease in viability, a G0/G1 cell cycle arrest, and an increase in apoptosis. The combination also resulted in decreased AML colony formation. We next explored potential mechanisms by which the combination treatment had a synergistic anti-leukemic effect. Both selumetinib and VTP-50469 treatment individually reduced phosphorylated ERK levels, but the combination had the most complete inhibitory effect. The combination treatment also resulted in loss of stem-like gene expression signatures, an increase in myeloid differentiation programs, and most strikingly, downregulation of targets of MYC signaling. Both MYC transcript and protein levels were reduced by each drug alone and even more effectively with the combination. Next, we evaluated the in vivo efficacy of this drug combination in three orthotopic pediatric AML and ALL pediatric PDX models harboring different KMT2A-r and RAS mutations (AML: KMT2A- AF10 with KRAS, KMT2A- PICALM with NRAS, ALL: MLL- AF4 with NRAS). NSG mice engrafted with PDX models via tail vein injection were assigned to four treatment groups: vehicle, chow containing revumenib (0.03%), selumetinib (50 mg/kg/day, 5 days a week), or the combination. After treatment for 18-28 days leukemia burden in bone marrow, spleen and peripheral blood was markedly decreased with the combination compared to the single drug-treated cohorts in 2 models with RAS mutations. In one of the AML models, single agent treatment with revumenib or selumetinib was highly efficacious at eradicating disease at the measured time point. We also found a striking decrease in levels of MYC, P-ERK and MEIS1 by western immunoblotting, highlighting on-target activity of the drugs and supporting our in vitro data for a role for MYC in the synergistic effect observed with the combination. In conclusion, new clinical data from a large cohort of COG patients demonstrates that KMT2A-r and co-occurring RAS pathway mutations portend a distinctively worse outcome for children with AML. The findings in our preclinical study suggest a promising, readily translatable targeted treatment for this patient population. Mechanistically, enhanced downregulation of both MYC signaling and the RAS/MAPK pathway with the combinatorial therapy is likely a strong contributor to the observed efficacy.

Relocalization of PU.1 Underlies the On- and Off-Tumor Effects of SWI/SNF Blockade in AML

SWI/SNF ATP-dependent chromatin remodeling activity has been identified as a strong dependency of acute myeloid leukemia (AML). In AML cell lines, genetic knockdown or chemical inhibition of the SWI/SNF ATPases SMARCA4 (BRG1) or SMARCA2 (BRM) by the selective small-molecule inhibitor BRM014 reduces proliferation and induces leukemic differentiation. These effects arise because SWI/SNF inhibition reduces DNA accessibility at binding sites of PU.1, a factor essential for driving high levels of MYC in leukemic cells. However, given that both SWI/SNF and PU.1 are essential in hematopoietic development, important questions regarding the use of SWI/SNF inhibitors against AML remain. First, the chromatin features that influence SWI/SNF-dependent PU.1 regulation within healthy hematologic and leukemic cells have not been established. Furthermore, the ability of SWI/SNF inhibitors to target leukemic cells within the bone marrow has remained uncertain, and the effects of these drugs in an immunocompetent setting have not been reported, despite the essential functions of SWI/SNF and PU.1 in normal hematopoiesis. Finally, the sensitivity of primary human AML samples and normal hematopoietic cells has also not been established. As a result, many questions regarding the nature of the therapeutic window for SWI/SNF inhibition against AML have remained unresolved. Here we evaluate SWI/SNF inhibition using BRM014 in clinically relevant models and examine the consequences of the deregulation of SWI/SNF-dependent PU.1 sites. We demonstrate that inhibition of SWI/SNF shifts PU.1 occupancy from enhancers to promoters in both leukemic and healthy hematopoietic cells. In AML, the shift of PU.1 away from enhancers to the promoters of differentiation-related genes results in loss of MYC expression and differentiation of leukemic cells. In non-leukemic mice, SWI/SNF inhibition suppresses PU.1-dependent B cell development and bone marrow-derived monocyte populations, reflecting off-tumor effects caused by SWI/SNF-dependent PU.1 relocalization. These effects influence peripheral immune cells, as well lineage-committed hematopoietic progenitors within the bone marrow. Moreover, we observe profound therapeutic response to SWI/SNF inhibition in an immunocompetent AML mouse model (Figure 1). In vivo, BRM014 induces differentiation of peripheral leukemic blasts and reduces the leukemic stem cell burden in bone marrow but also induces lymphocytopenia due to its off-tumor effects on PU.1. Nevertheless, the robust regression of leukemic burden seen in vivo over a short two-week treatment period provides a compelling justification for continued study of SWI/SNF inhibitors in the treatment of AML. Further, response of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and AML specimens highlight that normal hematopoiesis is affected at concentrations similar to those needed for therapeutic response in AML (Figure 2). Hence, hematopoietic and immune-related side effects, particularly at higher doses and over the duration of treatment, will be important considerations for clinical use of SWI/SNF inhibitors against AML and other tumors. Overall, our results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hexamethylene Amiloride Sensitizes AML to Venetoclax through Inducing Lysosomal Membrane Permeabilization

Chemotherapy remains the standard therapeutic approach for acute myeloid leukemia (AML). Although several new drugs have received approval for clinical usage, including the selective BCL2 inhibitor venetoclax, refractory disease or resistance continues to be a recurring theme, and exploring more effective therapies for AML treatment remains necessary. Intracellular pH dynamics are vital determinants for cancer progression and chemoresistance. Tumors maintain a more alkaline intracellular pH (pHi) dependent on proton exporters, including sodium-hydrogen exchangers (NHEs). Elevated NHE1 expression has emerged as a biomarker for the tumorigenesis and prognosis of solid tumors. However, whether targeting NHE1 with inhibitors could inhibit AML cell growth and the underlying mechanisms remain unknown. We found that NHE1 was highly expressed in AML and predicted a worse prognosis based on data analysis of public database. Moreover, NHE1 knockdown inhibited the growth of AML cells. In addition, NHE1 inhibitor Hexamethylene amiloride (HA) significantly inhibited proliferation and promoted apoptosis in a concentration-dependent manner in both AML cell lines and primary AML cells. Next, we combined HA with several first-line or novel drugs and found that HA combined with venetoclax significantly inhibited viability of AML cells. AML patients with higher NHE1 expression were less sensitive to BCL-2 inhibitors. This suggested that HA and venetoclax might be an effective combination to treat AML. As expected, HA synergized with venetoclax to inhibit proliferation and promote apoptosis. In vivo potential of HA combined with venetoclax was evaluated in a MV4-11-derived xenograft mouse model (Fig.1). The combinational regimen prolonged survival and alleviated leukemia burden in bone marrow, spleen and peripheral blood. We further validated the effect of HA with venetoclax with primary AML cells. Consistent with results in cell lines, HA combined with venetoclax also exerted synergistic effect in inhibiting viability and promoting apoptosis. RNA-Seq analysis revealed that lysosome pathway was significantly enriched in the combination group. We further verified the increased quantity of lysosome, and the swelling and rupture of lysosome with TEM (Fig.2). Moreover, HA combined with venetoclax induced obvious LGALS3 puncta formation in this cell line, which represented the occurrence of lysosomal membrane permeabilization (LMP), leading to the release of proteases which eventually results in cell death. In conclusion, our results for the first time provide evidence that the NHE1 inhibitor HA or its combination with venetoclax efficiently inhibits the growth of AML in vitro and in vivo. This study might shed light on the clinical treatment of AML involving proton exporter inhibitors with venetoclax as combinatory regime. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Establishment and Characterization of Mouse Models with Primary and Acquired BTK Inhibitor Resistance

Background: The second-generation Bruton's tyrosine kinase (BTK) inhibitor acalabrutinib is effective for treatment of patients with chronic lymphocytic leukemia (CLL), including high-risk and relapsed patients. Resistance to BTK inhibitors can develop not only due to on-target mutations, but also by bypassing the BTK signaling pathway. Mouse models represent useful tools in studying mechanisms of resistance as well as testing the efficacy of alternative therapy options to overcome resistance. Eµ-TCL1-transgenic (TCL1-tg) mice have the advantage of immunocompetence and resemble the human CD49d-high CLL cohort (Szenes et al., 2020). Transplantation of tumors from these mice to wild-type recipients allows acceleration of pathophysiology, selection of specific tumor clones, and more efficient testing of drug combinations. Here, we developed acalabrutinib resistance models by serial transplantation and long-term administration of the drug in drinking water, characterized these models, and compared molecular resistance signatures with those of human patient samples. Methods: Models were developed starting from four primary TCL1-tg mice at overt disease with tumor burden in spleen ranging up to 95% of lymphocytes. Serial retransplantation to wild-type C57BL/6J recipients for two to four rounds and acalabrutinib therapy pressure resulted in several drug resistant pedigrees. Mice were sacrificed at end-stage disease and tumor burden in spleen, bone marrow and blood was analyzed by flow cytometry using anti-CD5/CD19 antibodies. Purified leukemic cells were subjected to RNA-seq, real-time quantitative PCR and mutational analyses. Results: Three response patterns were identified in recipient mice: a) sustained sensitivity (models were stopped after 23 weeks of treatment), b) acquired resistance (with initial response followed by development of resistance under continuous therapy pressure and c) retransplantation-induced resistance in a case of an aggressive tumor clone that was responding to acalabrutinib in the parental mouse. The sensitive models displayed a 63% reduction in spleen weight upon treatment and a concomitant 65% reduction in blood tumor burden. In acquired or primary resistant mice, spleen weight and tumor burden was not significantly different from that of untreated mice, with blood tumor burden in the magnitude of 100,000 leukemic cells/µl and spleen tumor burden of 500-1000 million leukemic cells. Resistance could not be attributed to the mutational landscape of BTK and PLCγ2. Mutations in p53, Myc, Notch1, Atm, and Sf3b1 were detected but found to be intrinsic in individual ancestors and further present in the follow-up transplantations. RNA-seq analysis revealed 863 differentially expressed genes in mice with acquired resistance to acalabrutinib (compared to untreated, Figure 1) and 114 shared differentially expressed genes in mice with acquired or transplantation-based resistance. Gene set enrichment analysis demonstrated the presence of MAPK signaling and NF-kB signaling gene signatures within the resistant tumors. In particular, acquired resistance was accompanied by changes in the B cell receptor downstream components. We also compared the murine 863 gene signature to human samples from acalabrutinib resistant patients (progression on drug versus baseline) and found 13 genes are differently expressed in both, human and mouse. Validation for this gene expression data is ongoing and updated results will be presented. Conclusion: We have developed novel TCL1-tg based murine models of acalabrutinib resistance. Our models provide well-characterized tools to study primary as well as acquired BTK inhibitor resistance and allow the current testing of potential new therapeutic approaches to overcome resistance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effectiveness of an O-Alkyl Hydroxamate in Dogs with Naturally Acquired Canine Leishmaniosis: An Exploratory Clinical Trial.

Simple SummaryCanine leishmaniosis is a challenge in veterinary medicine and no drug to date has achieved parasite clearance in dogs. We have recently found that Vorinostat derivatives (O-alkyl hydroxamates) are active against Leishmania infantum intracellular forms and effective in laboratory models of visceral leishmaniasis without producing toxicity. We designed a clinical trial with 18 dogs naturally infected with the parasite and demonstrated that our flagship compound, MTC-305, is superior to the current first-line treatment of canine leishmaniasis, meglumine antimoniate, at reducing the parasite numbers in target organs (bone marrow, lymph nodes and blood) and improving the dogs’ clinical state. MTC-305 was not toxic in dogs, unlike the standard treatment that causes gastrointestinal alterations. We believe that, despite the limitations of the present work, MTC-305 and other O-alkyl hydroxamates are promising drugs in the fight against this neglected disease.Canine leishmaniosis is a challenge in veterinary medicine and no drug to date has achieved parasite clearance in dogs. Histone deacetylase inhibitors are a drug class widely used in cancer chemotherapy. We have successfully used O-alkyl hydroxamates (vorinostat derivatives) in the treatment of a laboratory model of visceral leishmaniasis without showing toxicity. In order to test the effectiveness of a particular compound, MTC-305, a parallel-group, randomized, single-centre, exploratory study was designed in naturally infected dogs. In this clinical trial, 18 dogs were allocated into 3 groups and were treated with either meglumine antimoniate (104 mg SbV/kg), MTC-305 (3.75 mg/kg) or a combination of both using a lower MTC-305 dose (1.5 mg/kg) through a subcutaneous route for 2 treatment courses of 30 days, separated by a 30-day rest period. After treatment, a follow-up time of 4 months was established. Parasite burden in bone marrow, lymph node and peripheral blood were quantified through qPCR. Antibody titres were determined through an immunofluorescence antibody test, and cytokine expression values were calculated through RT-qPCR. Treatment safety was evaluated through the assessment of haematological and biochemical parameters in blood, weight, and gastrointestinal alterations. Assessment was carried out before, between and after treatment series. Treatment with MTC-305 was effective at reducing parasite burdens and improving the animals’ clinical picture. Dogs treated with this compound did not present significant toxicity signs. These results were superior to those obtained using the reference drug, meglumine antimoniate, in monotherapy. These results would support a broader clinical trial, optimised dosage, and an expanded follow-up stage to confirm the efficacy of this drug.

GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.

CD38-Directed Vincristine Nanotherapy for Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although intensive chemotherapy greatly improved the survival rate, it is often accompanied by severe and lifelong side effects as a result of weak ALL selectivity. The intensive and poorly selective chemotherapy is also detrimental to patients' immune system. There is an urgent need to develop more selective and less toxic chemotherapy for ALL. Here, we report daratumumab-polymersome-vincristine (DP-VCR) as a CD38-directed nanotherapy for ALL. DP-VCR showed selective uptake in CD38-positive 697 and Nalm-6-Luc ALL cells and potent anti-ALL activity with an IC50 as low as 0.06 nM VCR, which was 13.7-fold more potent than free VCR. In contrast, no toxicity to human peripheral blood mononuclear cells was detected for DP-VCR even at 108.3 nM VCR. The apoptotic assays confirmed a high selectivity of DP-VCR to CD38-positive ALL cells. DP-VCR exhibited superior treatment of both 697 and Nalm-6-Luc orthotopic ALL models to all controls, as revealed by significant survival benefit and marked reduction of leukemia burden in bone marrow, blood, spleen, and liver. Importantly, DP-VCR induced few side effects. DP-VCR emerges as a safe and potent nanotherapy for CD38-positive ALL.

A Machine Learning Model Based on Tumor and Immune Biomarkers to Predict Undetectable Measurable Residual Disease (MRD) in Transplant-Eligible Multiple Myeloma (MM)

A Machine Learning Model Based on Tumor and Immune Biomarkers to Predict Undetectable Measurable Residual Disease (MRD) in Transplant-Eligible Multiple Myeloma (MM)

Circulating Tumor Cells (CTCs) in Smoldering and Active Multiple Myeloma (MM): Mechanism of Egression, Clinical Significance and Therapeutic Endpoints

Circulating Tumor Cells (CTCs) in Smoldering and Active Multiple Myeloma (MM): Mechanism of Egression, Clinical Significance and Therapeutic Endpoints

Enasidenib in Combination with Venetoclax in IDH2-Mutated Myeloid Malignancies: Preliminary Results of the Phase Ib/II Enaven-AML Trial

Enasidenib in Combination with Venetoclax in IDH2-Mutated Myeloid Malignancies: Preliminary Results of the Phase Ib/II Enaven-AML Trial

Deficiency of the E3 Ligase Hoip Impairs Adult Hematopoiesis and Myeloid Leukemia

Ubiquitination is a post-translational modification that plays important roles in the regulation of various cellular processes. The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase, which can specifically generate linear ubiquitin chains. LUBAC is composed of three subunits: catalytic Hoip (Rnf31), Hoil-1l (RBCK1), and Sharpin. LUBAC regulates canonical NF-κB signaling pathway and apoptosis. Several studies have shown that LUBAC is required for fetal hematopoiesis and tumorigenesis in several solid cancers, but the role of Hoip, which is the catalytic component of the LUBAC, in adult hematopoiesis and myeloid leukemia is unclear.To address whether Hoip is required for adult hematopoiesis, we used tamoxifen induced Hoip deleted mice (Hoip fl/fl; Rosa26-CreERT). Deletion of Hoip reduced the numbers of almost all cell fractions in bone marrow and peripheral blood, except for long-term hematopoietic stem cell (LT-HSC) fraction (CD34 - Flk2 - Lineage - c-Kit + Sca-1 +: LSK). Deletion of Hoip treated with tamoxifen markedly impaired colony formation of LT-HSCs in vitro. Among competitive transplantation assay using bone marrow cells from Hoip fl/fl; Rosa26-CreERT mice, deletion of Hoip treated with tamoxifen following stable reconstitution 8 weeks after transplantation led to a significant reduction of Hoip-deficient chimerism compared to control in vivo (Figure A). Deletion of Hoip induced apoptosis in bone marrow cells and increased the frequency of CD34 - LSK cells in S/G2/M phase of the cell cycle. Collectively, these data indicated that Hoip is required for adult hematopoiesis.To evaluate the consequences of Hoip deletion in myeloid leukemia propagation and maintenance, we utilized aggressive murine myeloid leukemia models driven by retroviral transduction of oncogenes: MLL-AF9 and NRAS G12V-driven acute myeloid leukemia (AML) and BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) models. Deletion of Hoip in established leukemia cells treated with tamoxifen markedly impaired colony formation of leukemia cells in vitro and led to a significantly longer survival with reduced disease burden in bone marrow and spleen in vivo in both of leukemic models (Figure B,C). Deletion of Hoip also induced apoptosis in leukemia stem cells (LSCs: c-Kit + Lineage - cells) (Figure D) and increased the frequency of LSCs in S-phase of the cell cycle. Furthermore, we evaluated the contribution of HOIP to human myeloid leukemia. Knockdown of HOIP by small hairpin RNA using lentivirus in several myeloid leukemia cell lines (THP-1, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro, and increased apoptosis. LUBAC inhibitor gliotoxin impaired the growth of murine and human leukemia cells. These results indicate that Hoip is essential for propagation and maintenance in murine and human myeloid leukemia.Because LUBAC regulates canonical NF-κB signaling pathway, we evaluated protein levels of p65 phosphorylation on Ser536 (p-p65) and IκBα in leukemia cells by flow cytometric analysis. Reduction in p-p65 and inhibition of IκBα degradation were observed in Hoip-deficient leukemia cells (Figure E), indicating that Hoip plays an important role in regulation of NF-κB signaling in myeloid leukemia.Since Hoip is the central component of LUBAC and the only catalytically active subunit of E3 ubiquitin ligase, we examined the importance of each domain of Hoip for myeloid leukemia propagation using Hoip mutant isoforms. Loss of colony-forming ability of Hoip-deficient AML cells could not be rescued by retroviral transduction of the Hoip mutants with deletion of UBA domain (which is required for interaction with the other LUBAC subunits) and with deletion of RBR domain and with mutation in C879A (which lack ligase activity) (Figure F), indicating that LUBAC ligase activity and interaction with the other LUBAC subunits are critical for leukemia propagation.Thus, our data demonstrated that Hoip is essential for adult hematopoiesis and myeloid leukemia. Given that patients with myeloid leukemia have shown increased activity of NF-κB signaling, inhibition of LUBAC ligase activity could serve as a promising strategy for treating myeloid leukemia. [Display omitted] DisclosuresJimbo: Japan Society for the Promotion of Science: Research Funding. Ito: Japan Society for the Promotion of Science: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Iwama: Nissan Chemical Corporation: Research Funding. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Konuma: Japan Society for the Promotion of Science: Research Funding; The Japanese Society of Hematology: Research Funding; SGH Foundation: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.