Leukemia Xenograft Mouse Model Research Articles

Simultaneous inhibition of FLT3 and HDAC by novel 6-ethylpyrazine-2-Carboxamide derivatives provides therapeutic advantages in acute myelocytic leukemia

Synergetic inhibition of FMS-like tyrosine kinase 3 (FLT3) and histone deacetylase (HDAC) by small molecule chimera presents a promising therapeutic approach for acute myeloid leukemia (AML) with FLT3 mutations. In this study, we first observed that the combined use of FLT3 inhibitor gilteritinib and HDAC inhibitor vorinostat increased the survival rate of leukemia xenograft mouse model. Then, we employed a pharmacophore fusion strategy to develop a novel series of FLT3/HDAC dual inhibitors. Among them, compound 25h demonstrated superior inhibitory activity against both FLT3 and HDAC. In particular, compound 25h exhibited enhanced anti-proliferation activity against MOLM-13 cells in comparison to gilteritinib, vorinostat, and their combination, while maintaining reduced cytotoxicity towards normal cells. Mechanistically, the heightened anti-tumor effect of compound 25h was attributed to its more potent regulation of intracellular pathways, notably phosphorylation of ERK, compared to single drug and combination treatments. Furthermore, compound 25h demonstrated superior anti-tumor efficacy in the MOLM-13 xenograft model compared to combination therapy, along with reduced in vivo toxicity. To conclude, we have identified a novel FLT3/HDAC dual inhibitor that could serve as a potential candidate for the treatment of AML.

A potent and selective ENL degrader suppresses oncogenic gene expression and leukemia progression.

The histone acylation reader eleven-nineteen leukemia (ENL) plays a pivotal role in sustaining oncogenesis in acute leukemias, particularly in mixed-lineage leukemia-rearranged (MLL-r) leukemia. ENL relies on its reader domain to recognize histone lysine acylation promoting oncogenic gene expression and leukemia progression. Here, we report the development of MS41, a highly potent and selective von Hippel-Lindau-recruiting ENL degrader that effectively inhibits the growth of ENL-dependent leukemia cells. MS41-induced ENL degradation reduces the chromatin occupancy of ENL-associated transcription elongation machinery, resulting in the suppression of key oncogenic gene expression programs and the activation of differentiation genes. MS41 is well-tolerated in vivo and substantially suppresses leukemia progression in a xenograft mouse model of MLL-r leukemia. Notably, MS41 also induces the degradation of mutant ENL proteins identified in Wilms' tumors. Our findings emphasize the therapeutic potential of pharmacological ENL degradation for treating ENL-dependent cancers, making MS41 not only a valuable chemical probe but also potential anticancer therapeutic for further development.

Cyclosporine A-resistant CAR-T cells mediate antitumour immunity in the presence of allogeneic cells

Chimeric antigen receptor (CAR)-T therapy requires autologous T lymphocytes from cancer patients, a process that is both costly and complex. Universal CAR-T cell treatment from allogeneic sources can overcome this limitation but is impeded by graft-versus-host disease (GvHD) and host versus-graft rejection (HvGR). Here, we introduce a mutated calcineurin subunit A (CNA) and a CD19-specific CAR into the T cell receptor α constant (TRAC) locus to generate cells that are resistant to the widely used immunosuppressant, cyclosporine A (CsA). These immunosuppressant-resistant universal (IRU) CAR-T cells display improved effector function in vitro and anti-tumour efficacy in a leukemia xenograft mouse model in the presence of CsA, compared with CAR-T cells carrying wild-type CNA. Moreover, IRU CAR-T cells retain effector function in vitro and in vivo in the presence of both allogeneic T cells and CsA. Lastly, CsA withdrawal restores HvGR, acting as a safety switch that can eliminate IRU CAR-T cells. These findings demonstrate the efficacy of CsA-resistant CAR-T cells as a universal, ‘off-the-shelf’ treatment option.

Bone Marrow Microenvironment-Responsive Nanomedicine Promotes Anti-Leukemia Activity In Vivo

Background: Leukemia escape and relapse is the leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). Work over the past decades has demonstrated that the CD47- signal-regulatory protein α (SIRPa) interaction mediated cancer cell immune escape. Although CD47-SIRPα interaction blockade contributes to cancer cell clearance by macrophages, how to deliver the anti-CD47 to bone marrow niche through an efficient drug delivery strategy and improve the cytotoxic activity of macrophages is an important issue that needs to be addressed. Methods: Here, we performed immunohistochemical (IHC) analysis and flow cytometry analysis of TIM-3 expression using samples of bone marrow from patients with or without relapse. In addition, according to the natural “myeloid niche homing” characteristic of AML cells and the low-oxygen microenvironment in bone marrow, we constructed β-cyclodextrin azobenzene linker (AZO-β-CD)-modified fusion protein and amantadine-modified HSA protein (HSA-ADA), followed by the addition of anti-CD47. Furthermore, the nanogel TIM3-targeting antibodies were modified on the surface of the nanogel to finally construct the supramolecular nanogel delivery system. Moreover, we detected the effect of target nanomedicine in a leukemia xenograft mouse model. Results: We demonstrated that in the bone marrow microenvironment of patients with relapse, the expression of TIM-3 significantly increased, especially in the CD34 +CD38 + leukemic progenitors, but not in the HSCs. Furthermore, we verified using nanomedicine could significantly increase the number of macrophages in the bone marrow. Importantly, the blockade of CD47-SIRPα signaling using nanomedicine markedly improved the anti-tumor activity in a leukemia xenograft mouse model. Conclusion: Thus, our findings reveal that nanomedicine could inhibit CD47-SIRPα signaling and contribute to anti-leukemia function effectively, which may be a new strategy for preventing and treating the recurrence of AML after transplantation.

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.

A Novel bispecific T-cell engager (BiTE) targeting CD22 and CD3 has both in vitro and in vivo activity and synergizes with blinatumomab in an acute lymphoblastic leukemia (ALL) tumor model

Immunotherapy has revolutionized cancer therapy. Two recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or chimeric antigen receptor T (CAR-T) cells. Blinatumomab, an FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating effector-target cell contact and T-cell activation that results in effective elimination of target B cells. Although CD19 is expressed by essentially all B-cell malignancies at clinical presentation, relapses with loss or reduction in CD19 surface expression are increasingly recognized as a cause of treatment failure. Therefore, there is a clear need to develop therapeutics for alternate targets. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments. Target binding of the anti-CD22 and anti-CD3 moieties was confirmed by flow cytometry. CD22-BiTE promoted in vitro cell-mediated cytotoxicity in a dose and effector: target (E:T)-dependent fashion. Additionally, in an established acute lymphoblastic leukemia (ALL) xenograft mouse model, CD22-BiTE demonstrated tumor growth inhibition, comparable to blinatumomab. Further, the combination of blinatumomab and CD22-BiTE yielded increased efficacy in vivo when compared to the single agents. In conclusion, we report here the development of a new BiTE with cytotoxic activity against CD22+ cells which could represent an alternate or complementary therapeutic option for B-cell malignancies.

Anti-tumor effect of antibody drug conjugate ASP1235 targeting Fms-like tyrosine kinase 3 with venetoclax plus azacitidine in an acute myeloid leukemia xenograft mouse model.

Antibody drug conjugates (ADC) are one of the attractive modalities for the treatment of acute myeloid leukemia (AML). Previously, we have developed ASP1235, a novel ADC targeting Fms-like tyrosine kinase 3 (FLT3) which is widely expressed on the leukemic blasts of AML patients. In this study, we sought to evaluate the therapeutic effect of ASP1235 in combination with venetoclax plus azacitidine, a novel standard-of-care treatment for elderly AML patients, in ASP1235 poor sensitive AML cells. To identify the suitable preclinical model, we first evaluated the growth inhibitory effect of ASP1235 on several leukemia cell lines expressing FLT3 and found that THP-1 cells were partially sensitive to ASP1235 in vitro. Furthermore, ASP1235 showed marginal anti-tumor activity in a THP-1 xenograft model. Compared to the leukemic blasts in most of the relapsed or refractory (R/R) AML patients tested, THP-1 cells expressed equivalent protein levels of Bcl-2, suggesting that ASP1235 in combination with venetoclax plus azacitidine is a rational treatment in the THP-1 model. In vitro, ASP1235 showed a cytotoxic effect on THP-1 cells in combination with venetoclax, and the combination effect was greater than the additive effect. Furthermore, ASP1235 also showed a combination effect with venetoclax plus azacitidine treatment. Similarly, the combination of ASP1235, venetoclax and azacitidine showed a superior anti-tumor effect in a THP-1 xenograft model without obvious body weight loss. These findings provide supportive evidence that the triple combination of ASP1235, venetoclax and azacitidine would improve the clinical outcome of ASP1235 monotherapy and venetoclax plus azacitidine regimen in AML patients.

A New in Vitro Model of Cortical Cerebral Organotypic Slices to Study Central Nervous System Microenvironment Modification By Leukemia Cells

Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy, accounting for 25% of all childhood cancers. Although great strides have been made in the treatment of childhood leukemia, close to 20% of patients will have resistant disease eventually leading to death. In approximately 40% of relapses, the central nervous system (CNS) is involved, alone or in combination with other sites, thus representing a major clinical concern. We recently demonstrated that ActivinA, a pleiotropic cytokine that belongs to the TGF-β superfamily, plays a crucial role within the leukemic niche. In addition, we showed by using a xenograft mouse model of human B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL), the ability of ActivinA to enhance both bone marrow (BM) engraftment and metastatic potential into extra-medullary sites of leukemic cells. Interestingly, an increased leukemic burden was observed in the CNS of mice receiving ActivinA-treated cells (Portale et al., 2019). In this study, we attempted to elucidate how leukemic NALM-6 cell line pre-treated or not with ActivinA modifies the CNS microenvironment by using an in vitro model of organotypic cortical brain slices. Methods:200-µm-thick brain slices from C57BL/6J or CX3CR1+/GFP (that express GFP in microglial cells) postnatal (P1-3) mice were obtained using a vibratome and placed onto a cell culture insert. After 7 days of recovery from cut, slices were microinjected with NALM-6 cells expressing iRFP670 pre-treated or not with ActivinA (50ng/mL for 24h). Proliferation of NALM-6 was quantified as the area of iRFP670 fluorescent signal in the brain slices up to 72h after injection. Brain slice cell death was measured by propidium iodide incorporation assay. Microglia activation was evaluated in CX3CR1+/GFP slices by longitudinally quantifying GFP signal up to 7 days post injection. In addition, microglia morphology was evaluated by shape descriptor parameters at 3 and 7days post-injection. Lastly, qRT-PCR assay to evaluate the modulation of microglial pro-and anti-inflammatory genes, was conducted. Results: We observed an exponential increase of iRFP670+ NALM-6 cells in the brain tissue up to 72h post-injection. Of note, pre-treatment with ActivinA significantly increase NALM-6 proliferation at 48 hours (p<0,05; n=7) and 72 hours (p<0,05; n=7) compared to not treated cells. ActivinA pre-treatment of NALM-6 cells was also associated with an higher mortality of brain slices compared to not stimulated NALM-6 cells at 24 hours (p<0,001; n=6 independent experiments) and 72 hours (p<0,05; n=6 independent experiments) after microinjection. By using CX3CR1+/GFP slices, we observed an increased microglia activation in ActivinA-pretreated NALM-6 microinjected slices compared to not treated at 5 (p<0,05; n=7) and 7 days (p<0.01; n=7) post-injection. Microglia morphological analysis showed that at 3 days post-injection, ActivinA pre-treated NALM-6 cells induced a slight increase of round-shaped microglia compared to CTRL (p<0,05; n=3) and to untreated NALM-6 injected slices (p<0,05; n=3), indicative of microglia reactivity to leukemic cells. However, at 7 days post-injection microglia from ActivinA pre-treated NALM-6 injected slices, showed an increase of cell area and perimeters (p<0,05; n=3), a reduced circularity and solidity (p<0,01; n=3) and a slight increase of aspect ratio (p<0,05; n=3) compared to CTRL slices, indicative of a reduced phagocytic activity of microglial cells. These modifications were associated with significant changes in gene expression, characterized by a down-regulation of microglial pro-inflammatory genes (iNOS, PAI-1; p<0.001; n=7) and an up-regulation of the anti-inflammatory ones (Arginase-1, CD206; p<0.01; n=7). Conclusions: We established an in vitro model to study leukemia cell interactions with brain tissue. Our data indicate that ActivinA pre-treatment promote higher NALM-6 proliferation in organotypic cortical brain slices compared to unstimulated leukemic cells. ActivinA pre-treated NALM-6 induce microglial modifications, characterized by a decrease reactivity and an induction of anti-inflammatory phenotype, favoring leukemic cell survival and proliferation. Our model can aid in answering important questions about brain changes after leukemic cell infiltration and could speed up the process of drug screening for leukemic patients.

Preclinical Evaluation of the Multiple Tyrosine Kinases Inhibitor Anlotinib in Leukemia Stem Cells.

Leukemia stem cells (LSCs) constitute the critical barrier to the cure of acute myeloid leukemia (AML) due to their chemoresistance and immune evasion property. Herein, the role of anlotinib, a multiple tyrosine kinase inhibitor, in killing LSCs and regulating chemoresistance and immune evasion was explored. Anlotinib treatment induced apoptosis of LSC-like cells as well as primary AML LSCs, while sparing the normal mononuclear cells in vitro. Moreover, anlotinib could impair the regeneration capacity of LSCs in the patient-derived leukemia xenograft mouse model. Mechanistically, anlotinib inhibited phosphorylation of c-kit, JAK2/STAT3, and STAT5, and downregulated STAT3 and STAT5 expression. In addition, anlotinib downregulated the anti-apoptotic proteins Bcl-2 and Bcl-xL, and upregulated Bax, thereby enhancing the sensitivity of LSCs to idarubicin in vitro. Intriguingly, anlotinib could also partially rescue the interferon-g production of T cells cocultured with LSCs by downregulating PD-L1 expression. In conclusion, anlotinib showed anti-LSC activity and the potential to enhance the sensitivity to idarubicin and inhibit the immunosuppressive feature of LSCs via JAK2/STAT signaling pathway downregulation in the preclinical study. Our results provided a rational basis for combinatory strategies involving anlotinib and chemotherapy or immunotherapy.

GARP-mediated active TGF-β1 induces bone marrow NK cell dysfunction in AML patients with early relapse post–allo-HSCT

AbstractRelapse is a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). However, the underlying mechanisms remain poorly understood. Natural killer (NK) cells play a crucial role in tumor surveillance and cancer immunotherapy, and NK cell dysfunction has been observed in various tumors. Here, we performed ex vivo experiments to systematically characterize the mechanisms underlying the dysfunction of bone marrow−derived NK (BMNK) cells isolated from AML patients experiencing early relapse after allo-HSCT. We demonstrated that higher levels of active transforming growth factor β1 (TGF-β1) were associated with impaired effector function of BMNK cells in these AML patients. TGF-β1 activation was induced by the overexpression of glycoprotein A repetitions predominant on the surface of CD4+ T cells. Active TGF-β1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Furthermore, pretreatment with the clinical stage TGF-β1 pathway inhibitor, galunisertib, significantly restored mTORC1 activity, mitochondrial homeostasis, and cytotoxicity. Importantly, the blockade of the TGF-β1 signaling improved the antitumor activity of NK cells in a leukemia xenograft mouse model. Thus, our findings reveal a mechanism explaining BMNK cell dysfunction and suggest that targeted inhibition of TGF-β1 signaling may represent a potential therapeutic intervention to improve outcomes in AML patients undergoing allo-HSCT or NK cell−based immunotherapy.

A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape.

CD73 catalyzes the conversion of ATP to adenosine, which is involved in various physiological and pathological processes, including tumor immune escape. Because CD73 expression and activity are particularly high on cancer cells and contribute to the immunosuppressive properties of the tumor environment, it is considered an attractive target molecule for specific cancer therapies. In line, several studies demonstrated that CD73 inhibition has a significant antitumor effect. However, complete blocking of CD73 activity can evoke autoimmune phenomena and adverse side effects. We developed a CD73-specific antibody, 22E6, that specifically inhibits the enzymatic activity of membrane-tethered CD73 present in high concentrations on cancer cells and cancer cell-derived extracellular vesicles but has no inhibitory effect on soluble CD73. Inhibition of CD73 on tumor cells with 22E6 resulted in multiple effects on tumor cells in vitro, including increased apoptosis and interference with chemoresistance. Intriguingly, in a xenograft mouse model of acute lymphocytic leukemia (ALL), 22E6 treatment resulted in an initial tumor growth delay in some animals, followed by a complete loss of CD73 expression on ALL cells in all 22E6 treated animals, indicating tumor immune escape. Taken together, 22E6 shows great potential for cancer therapy, favorably in combination with other drugs.

Targeting PI4KA sensitizes refractory leukemia to chemotherapy by modulating the ERK/AMPK/OXPHOS axis.

Background: The emergence of chemoresistance in leukemia markedly impedes chemotherapeutic efficacy and dictates poor prognosis. Recent evidence has revealed that phosphatidylinositol 4 kinase-IIIα (PI4KA) plays a critical role in tumorigenesis. However, the molecular mechanisms of PI4KA-regulated chemoresistance and leukemogenesis remain largely unknown.Methods: Liquid chromatography-mass spectrometry (LC-MS), patient samples and leukemia xenograft mouse models were used to investigate whether PI4KA was an effective target to overcome chemoresistance in leukemia. Enzyme-linked immunosorbent assay (ELISA) and molecular mechanics/generalized born surface area (MM/GBSA) method were employed to identify cepharanthine (CEP) as a novel PI4KA inhibitor.Results: High expression of PI4KA was observed in drug-resistant leukemia cells or in relapsed leukemia patients, which was correlated with poor overall survival. Depletion of PI4KA sensitized drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo by regulating ERK/AMPK/OXPHOS axis. We also identified cepharanthine (CEP) as a novel PI4KA inhibitor, which could undermine the stability of the PI4KA/TTC7/FAM126 complex, enhancing the sensitivity of drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo.Conclusions: Our study underscored the potential of therapeutic targeting of PI4KA to overcome chemoresistance in leukemia. A combination of the PI4KA inhibitor with classic chemotherapeutic agents could represent a novel therapeutic strategy for the treatment of refractory leukemia.

Targeting USP7 Suppresses Tumor Development By Promoting Ubiquitination of PHF8 and Suppressing SNAI1/Wnt Signaling in T-Cell Acute Lymphoblastic Leukemia

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy with a high unfavorable prognosis. Ubiquitin-specific-processing protease 7 (USP7) is one of the deubiquitinating enzymes attracting concentrated attention in current studies for cancers; it is also involved in regulation of oncogenic transcriptional program in T-ALL, and serves as a potential target to treat T-ALL. USP7 is interacted with PHD Finger protein 8 (PHF8), a histone lysine demethylase in T-ALL. However, the impact of the interaction in T-ALL development is undetermined. In this study, we explored the anti-tumor effect of targeting USP7 and its downstream substrate in T-ALL and underlying mechanism.MethodsIn vitro GST pull-down assay, co-immunoprecipitation assay, protein deubiquitination and stability assays were used to detect the interaction of USP7 with its direct substrate and the ubiquitination level. Gain-of-function(overexpression) and loss-of-function (lentiviral shRNA knockdown) approaches for USP7 were conducted for gene expression, CCK-8 cell proliferation assay, and apoptosis assay with annexin V + PI staining. ChIP-qPCR assay was used to explore the enrichment of histone markers in promoter region of target gene. In vivo human T-ALL xenograft mouse model was developed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse by tail vein injection of 2.5 × 10 6 /mouse and the leukemia engraftment was evaluated with human CD45+ cells by flow cytometry. The qPCR and western blot were carried out for detection mRNA level and protein level of related genes. The mRNA level of USP7 and its substrate PHF8 was examined by qPCR in 43 newly-diagnosed T-ALL patients with an approval of the Ethics Committee. Microarray datasets of T-ALL patients from GEO database were used to determine the USP7 and its substrate -related genes with web-based platform MEM and Limma.ResultsResults showed that USP7 directly interacted with PHF8; and overexpressed USP7 deubiquitinated and stabilized PHF8, and enhanced cell proliferation of JURKAT T-ALL cells. These data indicates targeting USP7 promotes cell proliferation of T-ALL cells through its substrate PHF8. Moreover, SNAI1 was identified as one of key co-expressed genes with PHF8 in microarray datasets from T-ALL patients, and knock-down of PHF8 suppressed the SNAL1 expression and increased the recruitment of repressive histone marker, H3K9me2 but decreased the enrichment of H3K4me3 in SNAI1 promoter. SNAI1 knockdown significantly induced the cell proliferation arrest and apoptosis of T-ALL cells (Fig.1A & 1B). With in vivo human leukemia xenograft mouse model, we further demonstrated that SNAI1 knockdown significantly reduce the spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity and also the inflammatory cell infiltration compared to the scramble shRNA control (Fig.1C-1F). Moreover, Wnt signaling was identified as SNAI1 interaction partners by the pathway analysis in the SNAI1 interaction genes in the microarray data from T-ALL patients. SNAI1 knockdown significantly suppressed the expression of the Axin2 and Survivn, the key components of Wnt signaling. These data suggested oncogenic role of USP7/PHF8/SNAI1/Wnt signaling in T-ALL. Next, we explored the effect of SNAI1 on USP7 knockdown-induced anti-tumor effect. We found that USP7 knockdown induced cell proliferation arrest and apoptosis, and overexpression of SNAI1 could block the effect in vitro. Furthermore, the in vivo data showed that USP7 knockdown significantly suppressed spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity, inflammatory cell infiltration, and the protein expression of PHF8, SNAI1, Axin2 and Survivin; and SNAI1 overexpression completely rescue the USP7 knockdown-mediated antitumor effect and restored the expression of Axin2 and Survivin in vivo (Fig.2).Conclusion:Our results demonstrated that targeting USP7 by shRNA induces the cell proliferation arrest and apoptosis by promoting ubiquitination of PHF8 and suppressing SNAI1/Wnt signaling in T-ALL. Our data also revealed the oncogenic roles of USP7/PHF8/SNAI1/Wnt signaling in T-ALL and suggested targeting the signaling pathway as potential therapy in T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rapamycin Pretreatment Rescues the Bone Marrow AML Cell Elimination Capacity of CAR-T Cells.

Ongoing clinical trials show limited efficacy for Chimeric antigen receptor (CAR) T treatment for acute myeloid leukemia (AML). The aim of this study was to identify potential causes of the reported limited efficacy from CAR-T therapies against AML. We generated CAR-T cells targeting Epithelial cell adhesion molecule (EpCAM) and evaluated their killing activity against AML cells. We examined the impacts of modulating mTORC1 and mTORC2 signaling in CAR-T cells in terms of CXCR4 levels. We examined the effects of a rapamycin pretreatment of EpCAM CAR-T cells (during ex vivo expansion) and assessed the in vivo antitumor efficacy of rapamycin-pretreated EpCAM CAR-T cells (including CXCR4 knockdown cells) and CD33 CAR-T cells in leukemia xenograft mouse models. EpCAM CAR-T exhibited killing activity against AML cells but failed to eliminate AML cells in bone marrow. Subsequent investigations revealed that aberrantly activated mTORC1 signaling in CAR-T cells results in decreased bone marrow infiltration and decreased the levels of the rapamycin target CXCR4. Attenuating mTORC1 activity with the rapamycin pretreatment increased the capacity of CAR-T cells to infiltrate bone marrow and enhanced the extent of bone marrow AML cell elimination in leukemia xenograft mouse models. CXCR4 knockdown experiments showed that CXCR4 contributes to the enhanced bone marrow infiltration capacity of EpCAM CAR-T cells and the observed reduction in bone marrow AML cells. Our study reveals a potential cause for the limited efficacy of CAR-T reported from current AML clinical trials and illustrates an easy-to-implement pretreatment strategy, which enhances the anti-AML efficacy of CAR-T cells.See related commentary by Maiti and Daver, p. 5739.

Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using cross-over dual-variable domain (CODV) format for acute myeloid leukemia (AML) treatment

ABSTRACT Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.

A Feasibility and Safety Study of a Novel CD19-Directed Synthetic T-Cell Receptor and Antigen Receptor (STAR) T-Cell Therapy for Refractory and Relapsed (R/R) B Cell Acute Lymphoblastic Leukemia (B-ALL)

Introduction Chimeric antigen receptor (CAR) T -cell therapy has demonstrated high response rates among patients with B cell malignancies yet remission durability and safety could be improved. We have developed a novel double-chain chimeric receptor Synthetic T Cell Receptor and Antigen Receptor (STAR) consisting of 2 protein modules each containing an antibody light or heavy chain variable region, the T Cell Receptor (TCR) a or b chain constant region fused to the OX-40 co-stimulatory domain, with the 2 modules linked by a self-cleaving Furin-p2A sequence that allows the modules to be proteolytically separated and reconstituted (Fig. 1A). Here, we report pre-clinical and first-in-human phase I trial results of CD19 STAR-T cell therapy for CD19+ R/R B-ALL. Methods Peripheral blood (PB) mononuclear cells were obtained from healthy donors and patients for the pre-clinical and clinical studies, respectively. T-cells were transduced with the STAR lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR T-cell antitumor function. For the clinical trial, from Dec. 2019 to Jun. 2020, 18 CD19+ R/R B-ALL patients (M/F 10:8) with a median age of 22.5 years (range: 6-68) were enrolled (NCT03953599). Patients received a conditioning regimen of IV fludarabine (25mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single STAR T-cell infusion. Once patients achieved complete remission (CR), they were given the option to proceed to consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT) or not. Results In preclinical studies, we found CD19 STAR T-cells to be superior to conventional CAR (BBz CAR) measured by the following parameters: 1) faster/stronger T-cell activation within 3 hours (76.67±2.621% vs 46.4±9.318%; p=0.0253); 2) higher cytokine production (4100.92±174.4 pg/ml vs 2556.78±563.39 pg/ml; p&amp;lt;0.05, Fig.1B) ;3) superior target killing ability (effector: target [E: T] ratio=1:1, 50.39±1.74% vs 60.85±1.52%, p&amp;lt;0.05. E:T ratio&amp;gt;1:1, p&amp;lt;0.01, Fig.1B); 4) robust elimination of B-ALL in a xenograft mouse model, where a lower E:T ratio was sufficient to eliminate an equal number of tumor cells (E:T ratio =1:1, STAR vs. BBz-CAR, p&amp;lt;0.01, Fig.1C). In the phase I trial, the median observation time was 69 (20-180) days. The median pre-treatment bone marrow (BM) blast level was 7.0% (0.1%-86.6%). All 18 patients received a single infusion of STAR T-cells at a median dose of 1×106/kg (5×105/kg-2.5×106/kg): low dose (5×105/kg) (n=3), medium dose (1×106/kg) (n=8) and high-dose (2-2.5×106/kg) (n=7). Three early enrollees subsequently received a second consolidation infusion of STAR T-cells at 1×106/kg (n=2) and 2×106/kg (n=1). The median STAR T-cell production time was 9 (7-13) days with a transduction efficacy of 57.4% (41.0%-78.2%). Two weeks post STAR T-cell infusion, 18/18 (100%) patients achieved CR with a negative minimal residual disease (MRD) status. After a median of 57 (43-66) days following STAR T-cell therapy, 8/18 patients made a choice to pursue consolidation allo-HSCT and all have remained in CR after a median follow-up of 110 (75-180) days. Of the 10 patients who did not undergo allo-HSCT, 1 relapsed on day 58 and died from relapse on day 63. This patient had a pre-CAR T-cell BM blast level of 86.6% with central nervous system leukemia. Another patient became MRD-positive with 0.09% blasts on day 30 per flow cytometry (FCM). The other 8 patients have remained in CR. Despite the achievement of a high CR rate, cytokine release syndrome (CRS) occurred only in 10/18 (55.6%) patients with 8 Grade I, and 2 Grade II CRS. Two patients developed Grade III neurotoxicity. After STAR T-cell infusion, CD19 STAR T-cells in PB were followed by qPCR and FCM. We saw high in vivo proliferation and persistence regardless of the infusion dose. The median peak level was reached on day 8.5 (day 4-10) with 4.9×104 (0.104-175×104) copy number/ug PB genomic DNA detectable at 6 months. Conclusion This study demonstrates the superiority of STAR T-cells compared to conventional CAR T-cells in terms of signaling capacity, cytokine production capability and anti-tumor potency in an animal model. The Phase I first-in-human study demonstrated technical feasibility, clinical safety and efficacy of STAR-T in treating CD19+ R/R B-ALL. A high CR could be achieved on day 14 with low toxicity. Longer-term observation of these patients and studies of larger patient cohorts are warranted. Disclosures No relevant conflicts of interest to declare.

ABL Genomic Editing Sufficiently Abolishes Oncogenesis of Human Chronic Myeloid Leukemia Cells In Vitro and In Vivo.

Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target the BCR-ABL junction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety of BCR-ABL junctions in CML patients, we utilized gene editing of the human ABL gene for clinical applications. Using the ABL gene-edited virus in K562 cells, we detected 41.2% indels in ABL sgRNA_2-infected cells. The ABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced the ABL gene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population in ABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with the ABL gene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using the ABL gene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of the ABL gene in maintaining CML cell survival and tumorigenicity in vitro and in vivo. ABL gene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.

Melatonin inhibits MLL-rearranged leukemia via RBFOX3/hTERT and NF-κB/COX-2 signaling pathways

MLL-rearranged leukemia is an aggressive malignancy associated with poor outcome, which is refractory to conventional treatment. Melatonin has been proven to exert anti-tumor activity, but the effect of melatonin on MLL-r leukemia and the underlying mechanism remain poorly understood. In this study, melatonin inhibited cell proliferation and induced apoptosis by activating the caspase-dependent apoptotic pathway in MLL-r leukemia cells. Mechanistic investigations revealed that melatonin suppressed the expression of hTERT by abrogating the binding activity of RBFOX3 to the hTERT promoter. Melatonin also blocked NF-κB nuclear translocation and suppressed NF-κB binding to the COX-2 promoter, thereby suppressing the expression of COX-2. In addition, clinical samples revealed that melatonin exerts anti-leukemic activity in primary MLL-r leukemia blasts ex vivo. In vivo, the mice treated with melatonin experienced a larger reduction in leukemic burden than the control group in a MLL-r leukemia xenograft mouse model. Collectively, these results suggest that melatonin inhibits MLL-rearranged leukemia through suppressing the RBFOX3/hTERT and NF-κB/COX-2 signaling pathways. Our findings provide new insights into the role of melatonin for MLL-r leukemia treatment.

Disruption of NOTCH1-MYC-CD44 Axis Targets Leukemia Initiating Cells (LIC) in T-ALL

Background:Persistence of leukemia initiating cells (LIC) in T acute lymphoblastic leukemia (T-ALL) results in relapse in 20- 25% of pediatric and over 50% of adult patients1. LIC are characterized by high CD44 expression and low reactive oxygen species (ROS) levels 2, 3. T-ALL LIC maintain low ROS by cystine/glutamate anti-porter complex of which CD44 is a key component 2, 4. CD44 also interacts with microenvironmental components; hyaluronic acid, osteopontin, fibronectin etc. NOTCH1 transcriptionally upregulates CD44/MYC by binding the upstream ‘super-enhancer’ sites. BRD4, a member of the bromodomain and extra terminal domain (BET) family, is a critical scaffold of super-enhancer complexes that binds acetylated histones (3 and 4) and drives NOTCH1 mediated MYC/CD44 transcription 5, 4, 1. We hypothesized that degradation of BRD4 with hetero-bifunctional PROteolysis TArgeting Chimera (PROTACTM) such as ARV-825, will lead to efficient and sustained downregulation of NOTCH1/MYC /CD44 transcription and disrupt cell intrinsic and extrinsic pathways for persistence of T-ALL LIC.Methods: We confirmed the anti-leukemia effect of ARV-825 (IC50 pico to low nanomolar) against T-ALL cells including early T-cell phenotype (ETP) and Gamma-secretase inhibitor (GSI) resistant T-ALL8. To confirm disruption of NOTCH1/MYC/CD44 axis in-vivo, we specifically tested the impact of BRD4 degradation on NOTCH1 and its target genes including MYC, CD44 and as functional readout, intra-cellular ROS, in a T-ALL/ patient-derived xenograft (PDX) mouse model of disseminated leukemia possessing a constitutively active NOTCH1 mutation. Mass cytometry based proteomic analysis (CyTOF) studies were done to quantitatively assess T-ALL LICs and suppression of NOTCH1-MYC-CD44 axis, and secondary transplantation was carried out into sub-lethally irradiated mouse recipients to functionally evaluate LIC elimination.Results: ARV-825 mediated sustained BRD4 degradation resulted in profound down-regulation of MYC, CD98, CD44 and its variants (CD44v). Moreover, we observed down-regulation of cell intrinsic anti-apoptotic proteins Bcl-2, Bcl-XL. As a functional correlate of down-regulation of CD98/CD44/CD44v (glutathione anti-porter system), flow cytometry confirmed increased intracellular ROS and decreased reduced glutathione (GSH).In a PDX mouse model of human T-ALL, ARV-825 treatment improved survival compared to mice treated with vehicle (P=0.002) (Fig.1). CyTOF analysis of mouse bone marrow cells showed quantitative reduction of phenotypically defined LIC (CD4+CD8+CD7+ CD19- ) 6,7 with down regulation of the NOTCH1-MYC-CD44 axis along with oncogenic molecules (transcription factors Myc and NFkB, cell cycle regulators, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins) in mice treated with ARV-825 (Fig.2). Finally, secondary transplantation of equal number of human CD45+ cells from Vehicle and ARV-825 treated mice in to NSG mice led to delayed leukemia development and extended survival of mice engrafted from ARV-825 treated mice (Vehicle:38 days Vs ARV-825: 58 days P=0.0001/ Vehicle:36.5 days Vs ARV-825: 50 days P=0.0001) (Fig.3), providing functional confirmation of LIC elimination.Conclusion: Degradation of BRD4 with PROTAC (ARV-825), modulates the NOTCH1/MYC/CD44 axis and has the potential of therapeutically targeting the LIC in T-ALL.ReferencePui CH, Carroll WL, Meshinchi S, Arceci RJ. Journal of clinical oncology 2011; 29(5): 551-565.Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H et al.Cancer cell 2011; 19(3): 387-400.Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN et al.Nature 2009; 458(7239): 780-783.Garcia-Peydro M, Fuentes P, Mosquera M, Garcia-Leon MJ, Alcain J, Rodriguez A et al.The Journal of clinical investigation 2018. doi: 10.1172/JCI92981Sanchez-Martin M, Ferrando A. Blood 2017; 129(9): 1124-1133.Cox CV, Martin HM, Kearns PR, Virgo P, Evely RS, Blair A. Blood 2007; 109(2): 674-682.Gerby B, Clappier E, Armstrong F, Deswarte C, Calvo J, Poglio S et al.Leukemia 2011; 25(8): 1249-1258.Sujan Piya, Hong Mu, Seemana Bhattacharya, Teresa McQueen, R. Eric Davis, Vivian Ruvolo, Natalia Baran, Yimin Qian, Craig M. Crews, M. James You , Patrick Zweider-McKay, Marina Konopleva, Hagop Kantarjian , Michael Andreeff1, Gautam Borthakur. 59 th Annual Meeting &Exposition, Atlanta GA: December 9-12, 2017. DisclosuresQian:Arvinas LLC Inc: Employment. Raina:Arvinas LLC Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Research Funding.

A novel miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry contributes to leukemogenesis in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies due to sophisticated genetic mutations and epigenetic dysregulation. MicroRNAs (miRNAs), a class of small non-coding RNAs, are important regulators of gene expression in all biological processes, including leukemogenesis. Recently, miR-375 has been reported to be a suppressive miRNA in multiple types of cancers, but its underlying anti-leukemia activity in AML is largely unknown.MethodsQuantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and HOXB3 in leukemic cells and normal controls. Targets of miR-375 were confirmed by western blot and luciferase assay. Phenotypic effects of miR-375 overexpression and HOXB3 knockdown were assessed using viability (trypan blue exclusion assay), colony formation/replating, as well as tumor xenograft assays in vivo.ResultsThe expression of miR-375 was substantially decreased in leukemic cell lines and primary AML blasts compared with normal controls, because DNA hypermethylation of precursor-miR-375 (pre-miR-375) promoter was discovered in leukemic cells but not in normal controls. Lower expression of miR-375 predicted poor outcome in AML patients. Furthermore, forced expression of miR-375 not only decreased proliferation and colony formation in leukemic cells but also reduced xenograft tumor size and prolonged the survival time in a leukemia xenograft mouse model. Mechanistically, overexpression of miR-375 reduced HOXB3 expression and repressed the activity of a luciferase reporter through binding 3′-untranslated regions (3’-UTR) of HOXB3 mRNA. Overexpression of HOXB3 partially blocked miR-375-induced arrest of proliferation and reduction of colony number, suggesting that HOXB3 plays an important role in miR-375-induced anti-leukemia activity. Knockdown of HOXB3 by short hairpin RNAs reduced the expression of cell division cycle associated 3 (CDCA3), which decreased cell proliferation. Furthermore, HOXB3 induced DNA methyltransferase 3B (DNMT3B) expression to bind in the pre-miR-375 promoter and enhanced DNA hypermethylation of pre-miR-375, leading to the lower expression of miR-375.ConclusionsCollectively, we have identified a miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry which contributes to leukemogenesis and suggests a therapeutic strategy of restoring miR-375 expression in AML.