Turing miRNA into infinite coordination supermolecule: a general and enabling nanoengineering strategy for resurrecting nuclear acid therapeutics

Background Clinical translation of therapeutic nuclear acid, particularly those targeting tumor progression, has been hampered by the intrinsic weaknesses of nuclear acid therapeutic including poor systemic stability, rapid clearance, low membrane permeability and lack of targeting ability. Small nuclear acid engineered into carrier-free nanodrugs with structural stability and disease targeting may be viable to overcome pharmaceutical obstacles of nuclear acid. Methods A general method through a mild and simple chemistry was established to convert therapeutic miRNA into an infinite Auric-sulfhydryl coordination supramolecular miRNA termed IacsRNA with near-spherical nanostructure, high colloid as well as anti-hydrolysis stability and low macrophage uptakes. Results IacsRNA presented the increased half-life period in circulation and accumulation at tumor sites in comparison to normal miRNA. Moreover, Iacs-miR-30c showed no toxicity of viscera and sanguis system in the 5-time injection dosage of the treatment. More importantly, Iacs-miR-30c potently suppressed the Wnt signaling pathway in vitro and in vivo, and effectively sensitized both potency of 5-Fu in PDX model of colon cancer and Anti-PD1 in B16F10 homograft model of melanoma. Conclusion Collectively, this work amply confirmed the design of IacsRNA as a general and viable strategy of nano-pharmaceutic to concert flimsy therapeutic miRNA into potential drugs. Considering from a broader perspective, the miRNA-initiated infinite coordination self-assembly strategy has distinct advantages in resurrecting nuclear acid therapeutics, probably bringing new inspiration to RNA-derived therapeutics of a great variety of human diseases including cancer. Graphical Abstract

Novel patient-derived models of DSRCT enable validation of ERBB signaling as a potential therapeutic vulnerability

Desmoplastic small round cell tumor (DSRCT) is characterized by the t(11;22)(p13;q12) translocation, which fuses the transcriptional regulatory domain of EWSR1 with the DNA-binding domain of WT1, resulting in the oncogenic EWSR1-WT1 fusion protein. The paucity of DSRCT disease models has hampered pre-clinical therapeutic studies in this aggressive cancer. Here, we developed preclinical disease models and mined DSRCT expression profiles to identify genetic vulnerabilities that could be leveraged for new therapies. We describe four DSRCT cell lines and one patient-derived xenograft (PDX) model. Transcriptomic, proteomic and biochemical profiling showed evidence of activation of the ERBB pathway. Ectopic expression of EWSR1-WT1 resulted in upregulation of ERRB family ligands. Treatment of DSRCT cell lines with ERBB ligands resulted in activation of EGFR, ERBB2, ERK1/2 and AKT, and stimulation of cell growth. Antagonizing EGFR function with shRNAs, small molecule inhibitors (afatinib, neratinib), or an anti-EGFR antibody (cetuximab) inhibited proliferation of DSRCT cells. Finally, treatment of mice bearing DSRCT xenografts with a combination of cetuximab and afatinib significantly reduced tumor growth. These data provide a rationale for evaluating EGFR antagonists in patients with DSRCT.

Ribonucleotide Reductase Regulatory Subunit M2 as a Driver of Glioblastoma TMZ-Resistance through Modulation of dNTP Production

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies have examined primary and recurrent GBM tumors, but it is difficult to study tumor evolution during therapy where resistance develops. To investigate this, we performed an in vivo single-cell RNA sequencing screen in a patient-derived xenograft (PDX) model. Primary GBM was modeled by mice treated with DMSO control, recurrent GBM was modeled by mice treated with temozolomide (TMZ), and during therapy GBM was modeled by mice euthanized after two of five TMZ treatments. Our analysis revealed the cellular population present during therapy to be distinct from primary and recurrent GBM. We found the Ribonucleotide Reductase gene family to exhibit a unique signature in our data due to an observed subunit switch to favor RRM2 during therapy. GBM cells were shown to rely on RRM2 during therapy causing RRM2-knockdown (KD) cells to be TMZ-sensitive. Using targeted metabolomics, we found RRM2-KDs to produce less dGTP and dCTP than control cells in response to TMZ (p<0.0001). Supplementing RRM2-KDs with deoxycytidine and deoxyguanosine rescued TMZ-sensitivity, suggesting an RRM2-driven mechanism of chemoresistance, established by regulating the production of these nucleotides. In vivo, tumor-bearing mice treated with the RRM2-inhibitor, Triapine, in combination with TMZ, survived longer than mice treated with TMZ alone (p<0.01), indicating promising clinical opportunities in targeting RRM2. Our data present a novel understanding of RRM2 activity, and its alteration during therapeutic stress as response to TMZ-induced DNA damage.

An In Vivo CRISPR Screening Platform to Identify New Therapeutic Targets in AML

First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing. We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery. Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically. Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML. Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. Disclosures Dharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Synergistic Combination Activity of the Novel GSPT1 Degrader CC-90009 in Acute Myeloid Leukemia Models

Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982). Methods: A high-throughput cell viability screen was performed to identify synergistic partners of CC-90009. AML cell lines (HL-60, HNT-34, KG-1, ML-2, NOMO-1, MOLM-13, MV4-11, F-36P, OCI-AML2, OCI-AML3) were treated with CC-90009 in combination with &gt; 70 compounds, including standard anti-AML agents, tyrosine kinase inhibitors (TKIs), unfolded protein response inducers, transcription inhibitors, and epigenetic agents. MOLM-13 and MV4-11 are fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) cell lines. Hits were validated in a colony formation (CF) assay using primary AML cells and bone marrow mononuclear cells (BMMC) from healthy donors. Synergy of the combination partners with CC-90009 was further assessed in AML patient-derived xenograft (PDX) models. Synergy between the isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib and CC-90009 was evaluated in a TF-1 erythroleukemia cell line overexpressing IDH2 R140Q mutant, and an IDH2 R140Q PDX model, AM7577. Results: Our high-throughput combination screen revealed multiple TKIs, epigenetic agents, and pro-apoptotic agents as potential combination partners of CC-90009 in AML cell lines. FLT3 inhibitors, including sunitinib, pexidartinib, midostaurin, lestaurtinib, crenolanib, and gilteritinib, synergized with CC-90009 to reduce viability in FLT3-ITD AML cell lines MV4-11 and MOLM-13. Similarly, the B-cell lymphoma 2 (BCL2) inhibitor VEN potentiated CC-90009-induced apoptosis and accelerated cell-autonomous killing. Reduction in levels of MCL-1, an anti-apoptotic factor, by CC-90009 most likely contributed to the synergy with VEN. We prioritized the evaluation of FLT3, BCL2, and IDH2 inhibitors as partners of CC-90009. In CF assays, midostaurin enhanced the inhibitory effect of CC-90009 in primary AML cells, without augmenting the effect of CC-90009 in CD34+ BMMC from healthy donors. Similarly, VEN enhanced the reduction in CF by CC-90009 in AML patient-derived BMMC without exacerbating the decrease in CF by CC-90009 in BMMC from healthy donors. We characterized FLT3 inhibitor/CC-90009 and BCL2 inhibitor/AZA/CC-90009 combinations in a FLT3-ITD PDX murine model, PDX1. The FLT3 inhibitor quizartinib significantly prolonged survival when combined with CC-90009 compared with either agent alone (P &lt; 0.001). Similarly, VEN/AZA/CC-90009 combination markedly extended survival compared with single agents or VEN/AZA doublets (P &lt; 0.001). The synergy between CC-90009 and epigenetic modulators was validated and further characterized in customized cell differentiation assays. Addition of CC-90009 to enasidenib, a mutant IDH2 inhibitor, enhanced differentiation and killing of CD34+ stem and progenitor cells, and increased differentiated CD235a+ erythroblasts, compared with enasidenib alone in a TF-1 cell line overexpressing IDH2 R140Q. Enasidenib/CC-90009 combination treatment reduced CD45+ malignant populations and increased differentiated CD14+ cells, leading to significantly prolonged animal survival in an IDH2 R140Q PDX model, AM7577, compared with either agent alone (P &lt; 0.0001). Conclusion: Using a high-throughput combination screen, we identified rational combination partners that synergize with CC-90009 in in vitro and in vivo AML models. Collectively, these results support the clinical evaluation of CC-90009 in combination with FLT3, BCL2, and IDH2 inhibitors to further improve treatment outcomes for patients with AML. Disclosures Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yao: Bristol Myers Squibb: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months, Research Funding. Pace: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Wang: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flandin-Blety: Bristol Myers Squibb: Current Employment. Benitez: Bristol Myers Squibb: Current Employment. Guarinos: Bristol Myers Squibb: Current Employment. Hoffmann: Bristol Myers Squibb: Current Employment. Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Bcl2 Inhibitor and DNA Hypomethylating Agent Prolong Survival in Patient-Derived Xenograft Model of Down Syndrome Myeloid Leukemia By Synergistic Downregulation of Cytokine Signaling

Background: Down syndrome is known for its leukemia predisposition effects. Children with Down syndrome myeloid leukemia who fail chemotherapy, have a poor outcome. No targeted therapies are available for this population. We have generated and extensively characterized several patient-derived xenograft (PDX) models of Down syndrome AML (Barwe et al., 134:2683, Blood, 2019). We evaluated the efficacy of a combination of DNA hypomethylating agent, azacitidine and Bcl2 inhibitor, venetoclax in PDX model of Down syndrome AML. Although this drug combination is used in the clinic for the treatment of adult AML, their combinatorial mechanism of action is not well known. Methods: NTPL-386 (3 x 10 6 cells) were injected intravenously in NSG-SGM3 mice. Disease progression was monitored by determination of the percentage of human chimerism in peripheral blood by flow cytometry. Mice were randomly assigned to treatment groups (n=5) on day 13 when human CD45+ cells were detectable in blood. Azacitidine (2.5 mg/Kg) was administered intraperitoneally on days 13-19 and venetoclax (100 mg/Kg) was delivered orally on days 13-40 (Fig. 1A). Mice were monitored daily for pre-determined experimental endpoints and were euthanized when any of the endpoints were attained. Kaplan-Meier survival plots were generated. AML cells were harvested from treated mice and lysed, RNA was isolated and sequenced. Pathway analysis was performed to identify the mechanism of action of these two drugs in combination. Results: Azacitidine treatment for one week increased median survival by 8 days compared to untreated mice (Fig. 1B). Venetoclax treatment for 4 weeks was more effective than azacitidine and extended survival by 27 days (P&lt;0.005, compared to untreated). We observed that the combination of venetoclax and azacitidine was much more efficient and prolonged survival by 39 days in this PDX model of refractory Down syndrome AML (P&lt;0.005 and P&lt;0.05 compared to single agent azacitidine or venetoclax respectively). This combination performed better than chemotherapy consisting of cytarabine and daunorubicin, which showed a 11-day improvement in median survival compared to untreated in our previous study (Barwe et al., 134:2683, Blood, 2019). To understand the mechanism of synergism between these two drugs, we conducted transcriptome analysis on cells harvested from mice (n=3 per group) receiving venetoclax and azacitidine, alone or in combination. The log fold changes for each treatment with respect to untreated were calculated and filtered based on FDR&gt;0.05. The genes that were differentially regulated in the combination treatment (Fig. 1C, blue circle in the Venn diagram) were subjected to ToppGene analysis. A heatmap of the differentially regulated genes shows clustering of the replicates from individual mice (Fig. 1D). 'Hematopoietic cell lineage' and 'cytokine signaling in immune system' were the top modulated pathways in the combination treatment. A heatmap of the logFC of the differentially regulated genes from this pathway showed the modulation of these genes in the combination in comparison with individual drug treatment (Fig. 1E). The differentially regulated genes in the combination treatment that showed high variance from the range defined by individual drug treatment belonged to one of three categories - cell surface proteins, cytokines, and stem cell markers. These data indicate that the synergistic downregulation of cytokine signaling is likely responsible for the combinatorial effect of venetoclax and azacitidine. Conclusion: The combination of venetoclax and azacitidine was effective in prolonging survival in a highly refractory Down syndrome AML PDX model (NTPL-386) generated in the laboratory. The top genes differentially regulated by the combination treatment which varied the most in magnitude from the individual drug treatment identified genes that are likely to contribute to the synergism between the two drugs. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

SLC5A3 Transports Myo-Inositol to Support the Growth of Acute Myeloid Leukemia

Metabolic reprogramming contributes to tumor development and sustains cancer cell proliferation. Like other cancers, acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival, has altered metabolic features, providing new possibilities for AML treatment. Since the niche can reshape the metabolic properties of cancer cells, it is critical to validate AML metabolic vulnerabilities in a proper microenvironment. To this end, we optimized a protocol for CRISPR screening in orthotopic xenograft AML models, including patient-derived-xenograft (PDX) models tractable for CRISPR-editing, to enable the systematic evaluation of the physiological relevance of top AML dependencies. We performed in vivo screens in MV4-11 and U937 cell lines and a PDX model, which converged to reveal the sodium/myo-inositol cotransporter SLC5A3 as a top-ranked in vivo gene target. We have validated the SLC5A3 dependency in additional AML cell line and PDX models; SLC5A3 deletion consistently induced apoptotic cell death in all AML models tested. We also observed cell-context dependent alterations of cell cycle and differentiation. In addition, using a PDX model with a doxycycline-inducible CRISPR system, we have confirmed that induction of SLC5A3 knockout post transplantation significantly represses AML progression and prolonged mouse survival. Next, we investigated whether the growth defect caused by SLC5A3 knockout results from the myo-inositol deficiency. Augmentation of myo-inositol concentration in the standard culture medium completely rescued the proliferation of SLC5A3-knockout cells. In accordance, depletion of myo-inositol from the culture medium largely impeded the growth of parental AML cells, causing similar phenotypes as SLC5A3-deletion, with cell-context dependent alterations of cell cycle and induction of apoptotic cell death. Together, these data reveal that myo-inositol is a critical metabolite for AML. Since a subset of AML cell lines were not dependent on SLC5A3 based on a genome-scale CRISPR screen dataset (DepMap), we explored the potential biomarkers associated with SLC5A3 essentiality in AML. Intriguingly, the low expression of Inositol-3-phosphate synthase 1 (ISYNA1) predicted a strong SLC5A3 dependency in AML cell lines. In addition to importing myo-inositol from the extracellular fluid, cells can also synthesize myo-inositol de novo from glucose 6-phosphate, and ISYNA1 encodes the rate-limiting enzyme in this myo-inositol biosynthesis pathway. We confirmed low expression of ISYNA1 protein in AML cells sensitive to SLC5A3 deletion, and importantly, overexpression of ISYNA1 can completely relieve the SLC5A3 dependency. Moreover, knockout of ISYNA1 in an ISYNA1-high cell line M07e exacerbated the growth defect associated with SLC5A3 deletion. Altogether, these results strongly demonstrate that SLC5A3 becomes essential in AML cells with insufficient myo-inositol biosynthesis capacity to support AML proliferation. Finally, we have investigated the clinical features associated with low ISYNA1 expression in genomic datasets of primary AML samples, including TCGA and Beat AML, to postulate the patient population that can benefit from a SLC5A3-directed therapy. Interestingly, low ISYNA1 expression is associated with FAB M4 and M5 AML subtypes. In accordance, a monocyte lineage gene signature is enriched in ISYNA1-low samples. In addition, AML samples with low ISYNA1 expression tend to have IDH2 or DNMT3 mutations. Collectively, our study demonstrated that SLC5A3 is a strong metabolic dependency in AML, and targeting SLC5A3 can provide a therapeutic opportunity for a subset of monocytic AML. Disclosures Dharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: AstraZeneca: Consultancy; KronosBio: Consultancy; Bristol Myers Squibb: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

HDACi Targets IKZF1 Deletion High-Risk Acute Lymphoblastic Leukemia By Inducing IKZF1 Expression and Rescuing IKZF1 Function in Vitro and In Vivo

The IKZF1 gene codes the transcription factor IKAROS with key regulatory functions in lymphopoiesis. Recurrent focal IKZF1 deletions (IKZF1 del), which affects the coding regions of IKZF1, have been identified as poor outcome in 30-40% adult B-ALL. Loss of IKAROS confer stem cell-like phenotype, upregulation of self-renewal capacity and cell-adhesion molecules, and TKI resistance. However, there are not specifically therapeutic options for IKZF1 del ALL and current protocols could not abrogate the adverse effect of IKZF1 del.Considering that IKAROS acts as a key component of the nucleosome remodeling and deacetylation (NuRD) complex engaging in development and metabolism, we speculate that epigenetic drugs, such as HDACi, might play a potent role in IKZF1 del high-risk B-ALL. Firstly, B-ALL cell lines (IKZF1 del: MUTZ-5, MHH-CALL-4; IKZF1 wt: NALM6) and primary patient samples (n=10, 5 with IKZF1 del and 5 with IKZF1 wt) were treated with different HDACi, including valproic acid, vorinostat, romidepsin, RGFP966 and a novel HDAC-selective inhibitor tucidinostat. But noteworthily, only tucidinostat yielded specific and selective proliferation inhibition in IKZF1 del cell line(IC 50=1.377±0.05) and IKZF1 del patients samples (IC 50=2.318±0.07), compared with the effect on IKZF1 wt cells. Interestingly, tucidinostat induced remarked increase of mRNA and protein of IKZF1 expression in leukemia bulk and IKZF1 del single cell. Seahorse metabolic flux assay, lactate and ATP measurements was performed and revealed that tucidinostat treatment reduced glycolysis (P=0.0067), lactic acid (P＜0.0001) and ATP level (P＜0.0001) in IKZF1 del B-ALL cell lines. To verify metabolic change is depend on IKZF1 induction or not,dominant-negative Ikaros isoform 6 (DN-IK6), deletion of exons 4-7, was transfected into IKZF1 wt Nalm-6 cell line to negative regulate of IKZF1 wide-type expression. Overexpression of DN-IK6 in Nalm-6, increases sensitivity to tucidinostat, glycolytic capacity(p=0.05) and glycolytic reserve (p=0.012) also increases. While tucidinostat treating with the IK6-Nalm-6, tucidinostat would restore the transcriptional repressor function of the remaining wild-type IKZF1 allele and decrease glycolytic capacity(p=0.011) and glycolytic reserve(p=0.014). Notably, the metabolic rate-limiting enzymes HK2 and PKM2 were strongly repressed. These data indicate that tucidinostat reverses the metabolic reprogramming of glycolysis or Warburg effect in IKZF1 del B-ALL in an IKZF1-inducing dependent manner. For in vivo study, PDX model with immunodeficient NOD/SCID/IL2Rgnull mice were injected with heavily-treated refractory/relapsed IKZF1 del B-ALL patient samples (n=2) and treated with tucidinostat with different dosage of 5-12.5mg/kg/day. Administration of tucidinostat observed IKAROS expression trajectory and resulted in prolonged animal survival in IKZF1 del B-ALL PDX model（P＜0.0001). Secondary transplantation of ALL cells from tucidinostat or vehicle-treated (1 x10 6) recipients revealed significantly improved survival in tucidinostat -treated group (p= 0.0235). These results indicate that tucidinostat treatment might elimination leukemia-initiating cells.Additionally, to profile the IKZF1 del B-ALL chromatin accessibility changes after tucidinostat-treatment. We performed ATAC-seq and observed a clear increase in accessibility at TCA cycle related gene and decrease in accessibility at glycolysis related gene.Furthermore, tucidinostat, formerly known as chidamide, was added to an open-label, one-arm PDT-Ph-like-ALL trial targeting adult Ph-like ALL, which is characterized with high frequency in IKZF1 deletions (Clinicaltrials.gov. NCT03564470). Preliminary data of PDT-Ph-like-ALL indicate that tucidinostat was effective and well-tolerated, yielded promising response in IKZF1 del Ph-like ALL (ASH2018, poster 4011; EHA 2019, PF181). Collectively, our study demonstrates that the novel HDAC-selective inhibitor, tucidinostat, could specifically target IKZF1 del high-risk B-ALL, by restoring the IKZF1 expression, resulting in attenuation of proliferation, reverse the Warburg effect and improvement of the survival in PDX model and preliminary data in clinical trial. These findings provide mechanistic insights and a promising therapeutic strategy for IKZF1 haploinsufficiency alterations B-ALL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.