AML Cells Research Articles

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1)

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5′ and 3′UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

Synergistic cytotoxicity of fludarabine, clofarabine, busulfan, vorinostat and olaparib in AML cells

Combinations of nucleoside analog(s) and DNA alkylating agent(s) are used for cancer treatment as components of pre-transplant regimens used in hematopoietic stem cell transplantation. Their efficacies are enhanced by combining drugs with different mechanisms of action, which also allows a reduction in the individual drug dosages and thus potentially in toxicity to the patient. We hypothesized that addition of SAHA and olaparib, an HDAC- and a PARP-inhibitor, respectively, to the established combination of fludarabine, clofarabine and busulfan would enhance AML cell cytotoxicity. Exposure of the AML cell lines KBM3/Bu2506, MV4-11, MOLM14 and OCI-AML3 to the 5-drug combination resulted in synergistic cytotoxicity with combination indexes < 1. Increased protein acetylation and decreased poly(ADP-ribosyl)ation were observed, as expected. Activation of apoptosis was suggested by cleavage of Caspase 3 and PARP1, DNA fragmentation, increased reactive oxygen species, and decreased mitochondrial membrane potential. The reduction in poly(ADP-ribosyl)ation was independent of caspase activation. Several proteins involved in DNA damage response and repair were downregulated, which may be contributing factors for the observed synergism. The increased phosphorylation of DNAPKcs suggests inhibition of its kinase activity and diminution of its role in DNA repair. A similar synergism was observed in patient-derived cell samples. These findings will be important in designing clinical trials using these drug combinations as pre-transplant conditioning regimens for AML patients.

Discovery of Pyrazolo[1,5-a]pyrimidine Derivative as a Novel and Selective ALKBH5 Inhibitor for the Treatment of AML.

M6A (N6-methyladenosine) plays a significant role in regulating RNA processing, splicing, nucleation, translation, and stability. AlkB homologue 5 (ALKBH5) is an Fe(II)/2-oxoglutarate (2-OG)-dependent dioxygenase that demethylates mono- or dimethylated adenosines. ALKBH5 can be regarded as an oncogenic factor for various human cancers. However, the discovery of potent and selective ALKBH5 inhibitors remains a challenge. We identified DDO-2728 as a novel and selective inhibitor of ALKBH5 by structure-based virtual screening and optimization. DDO-2728 was not a 2-oxoglutarate analogue and could selectively inhibit the demethylase activity of ALKBH5 over FTO. DDO-2728 increased the abundance of m6A modifications in AML cells, reduced the mRNA stability of TACC3, and inhibited cell cycle progression. Furthermore, DDO-2728 significantly suppressed tumor growth in the MV4-11 xenograft mouse model and showed a favorable safety profile. Collectively, our results highlight the development of a selective probe for ALKBH5 that will pave the way for the further study of ALKBH5 targeting therapies.

DNMTs-mediated SOCS3 methylation promotes the occurrence and development of AML.

As a tumor suppressor gene, SOCS3 inhibits the growth of tumor cells by regulating JAK/STAT signaling pathway through negative feedback. This study aimed to investigate the biological function and mechanism of SOCS3 methylation mediated by DNMTs in the development of AML. Bone marrow samples were collected from 70 AML patients and 20 healthy volunteers. The expression and methylation status of each gene were detected by RT-qPCR, western blot and MS-PCR, and the growth and apoptosis rate of leukemia cell lines were detected by CCK-8 and flow cytometry. The effects of changes in SOCS3 gene expression and methylation status of AML cell lines were observed by gene transfection and gene knockdown. The methylation rate of SOCS3 in AML initial treatment group was significantly higher than that in the remission group and the normal control group (60% vs. 0%, 0%). The expression of SOCS3 in the SOCS3 methylation group was significantly lower than that in the non-methylated group and control group, while the expression of DNMT1, DNMT3a, p-JAK2, p-STAT3 and p-STAT5 were significantly higher than those in the non-methylated group and control group. Demethylation treatment, SOCS3 transfection and DNMT3a knockdown could up-regulate the expression of SOCS3, which decreased the proliferation and increased the apoptosis of leukemia cell lines. SOCS3 methylation mediated by DNMTs promotes the occurrence and development of AML and can be used as a potential biomarker for the diagnosis and efficacy evaluation of AML.

TIM-3/Gal-9 interaction affects glucose and lipid metabolism in acute myeloid leukemia cell lines.

T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in acute myeloid leukemia and master cell survival and proliferation. In this study, we aimed to explore the effect of TIM-3 interaction with its ligand galectin-9 (Gal-9) on glucose and lipid metabolism in AML cell lines. HL-60 and THP-1 cell lines, representing M3 and M5 AML subtypes, respectively, were cultured under appropriate conditions. The expression of TIM-3 on the cell surface was ascertained by flow cytometric assay. We used real-time PCR to examine the mRNA expression of GLUT-1, HK-2, PFKFB-3, G6PD, ACC-1, ATGL, and CPT-1A; colorimetric assays to measure the concentration of glucose, lactate, GSH, and the enzymatic activity of G6PD; MTT assay to determine cellular proliferation; and gas chromatography-mass spectrometry (GC-MS) to designate FFAs. We observed the significant upregulated expression of GLUT-1, HK-2, PFKFB-3, ACC-1, CPT-1A, and G6PD and the enzymatic activity of G6PD in a time-dependent manner in the presence of Gal-9 compared to the PMA and control groups in both HL-60 and THP-1 cell lines (p > 0.05). Moreover, the elevation of extracellular free fatty acids, glucose consumption, lactate release, the concentration of cellular glutathione (GSH) and cell proliferation were significantly higher in the presence of Gal-9 compared to the PMA and control groups in both cell lines (p < 0.05). TIM-3/Gal-9 ligation on AML cell lines results in aerobic glycolysis and altered lipid metabolism and also protects cells from oxidative stress, all in favor of leukemic cell survival and proliferation.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

FT538, iPSC-Derived NK Cells Are Potent Inducers of Apoptosis in AML Cells and Their Effect Is Synergistic in Combination with Approved Therapeutic Strategies

Background: Natural killer (NK) cell-based therapies have been increasingly studied with their advantages of less treatment-related toxicity and lack of HLA restriction. Induced pluripotent stem cell (iPSC)-derived NK cells offer an opportunity for a standardized, off-the-shelf treatment with the potential to treat a wider patient population. FT538 cells are triple gene-edited iPSC-derived NKs that express a high affinity non-cleavable CD16 maintaining constant activity, a CD38 knock out preventing CD38 antibody-induced fratricide, and an IL-15/IL-15R fusion preventing the need for cytokine administration, the main source of side effects in NK cell-based therapies. Several studies have shown success in treating AML with NK cell therapy, but the effect of iPSC-derived NK cell therapy in AML is unexplored. We hypothesized that FT538 cells may be able to target high-risk AML that is traditionally resistant to most chemotherapeutic agents and synergize with Venetoclax, 5-azacytidine, Cytarabine, and Gilteritinib. Methods: To examine the effect of the FT538 cells on AML cells, we co-cultured FT538 cells at ratios of 2:1, 4:1, and 8:1 with 8 AML cell lines: OCI-AML2, OCI-AML3, THP1, U937, Molm-13, Molm-14, MV4-11, and Kasumi-1, and with cells from 11 different AML patients. We analyzed the NK cell-induced apoptosis through an Annexin-V binding assay using IncuCyte live cell imaging. As Molm-13 and MV4-11 cell lines are FLT3 mutated, to determine the combination effect of FLT3 inhibitors with FT538 cells, we treated Molm-13 and MV4-11 cells with Gilteritinib for 24 hours before seeding the FT538 cells and co-culturing overnight. The effects of the combination were then analyzed through flow cytometry with a Beckman Coulter CytoFlex. Finally, to determine the combinatorial effect of the FT538 cells with approved therapies, we treated OCI-AML3 and U937 cell lines with Venetoclax, Azacitidine, and Cytarabine. After treatment, we performed flow cytometry to measure the leukemic cell death following the combinatorial therapies. Results: We observed FT538 NK-mediated killing in all the leukemic cell lines tested. A two-way ANOVA determined that an increase in the FT538 to AML cell ratio resulted in an increase in NK cell-mediated killing of AML cells (p&amp;lt;0.0001). Interestingly, the number of apoptotic AML cells was 1.5 to 6.1-fold higher in the OCI-AML2, OCI-AML3, U937, and THP1 cell lines than in the Molm-13, Molm-14, MV4-11, and Kasumi-1 cell lines (p&amp;lt;0.0001). This could be related to the FLT3 mutations in the Molm-13, Molm-14, and MV4-11 cell lines or to the TP53 mutation in the Kasumi-1 cell line. Further, to validate this finding in primary AML cells, we observed a ratio-dependent effect on FT538-mediated killing of AML cells. However, we did not observe any statistical difference by mutation, though this could be related to the limited sample size for specific mutations. Finally, flow cytometric analysis of the effects of combination treatment demonstrated that the FT538 cells synergize with Venetoclax, or Cytarabine, or Azacitidine, or Gilteritinib (p&amp;lt;0.05) in the killing of AML cells. This could be because NKG2DL upregulation has been shown in AML cells treated with Venetoclax and FLT3 inhibitors and in Cytarabine-resistant AML cells, and the NKG2D/NKG2DL pathway is a main mechanism of NK cell-mediated killing. Conclusion: iPSC-derived NK therapy offers a standardized, off-the-shelf option for NK cell therapies. FT538 iPSC-derived NK cells induce apoptosis in AML cell lines and patient samples in a dose-dependent manner and show a synergistic effect with Venetoclax, Gilteritinib, Azacitidine, and Cytarabine. Therefore, iPSC-derived NK cell therapy may be a promising possibility for AML treatment, particularly for patients resistant to standard therapies.

Regulation of Metabolic Homeostasis By TRAF6 Contributes to the Leukemia Progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that mediates innate immune signaling that converges on key mediators of Toll-like receptor and interleukin-1 signaling. Dysregulated TRAF6 expression and innate immune pathway activation is implicated in the pathogenesis of various myeloid malignancies. For instance, increased TRAF6-mediated signaling in hematopoietic stem and progenitor cells (HSPCs) results in myelodysplastic syndrome-like cellular features, such as the functional impairment of HSPCs. On the other hand, our previous study indicated that loss of TRAF6 induces the leukemic transformation of pre-leukemic cells ( Muto et al. Cell Stem Cell). These paradoxical observations suggests that TRAF6 acts as both an oncogene and tumor suppressor gene in hematopoietic cells in a disease type- and context-dependent manner. However, the direct consequence of TRAF6 loss in AML cells, rather than in pre-leukemic cells, has not yet been explored. Therefore, the role of TRAF6 in the pathogenesis of AML remains obscure. In this study, we found elevated level of TRAF6 expression in AML cells, and determined the effect of TRAF6 loss on the cellular and molecular features of AML cells. We proposed a novel role of TRAF6 as a regulator of metabolic homeostasis, which is important for the progression of AML. To determine the requirement of TRAF6 in AML, we first evaluated cell the effect of TRAF6 loss in AML cell lines on proliferative capacities. Multiple types of human AML cell lines showed significant growth impairment upon TRAF6-knockdown. Furthermore, transplantation of TRAF6-deficient HSPCs expressing the leukemic oncogene MLL-AF9 to the recipient mice resulted in suppression of overt leukemia. These findings confirm that loss of TRAF6 suppresses leukemic cell function in vitro and in vivo. To assess the molecular mechanism of inhibitory effect of TRAF6 loss in AML, we next performed gene expression analysis using human AML database and TRAF6-knockdown AML cell lines. Interestingly, we found that reduced levels of TRAF6 in AML is associated with changes in the expression of genes involved in mitochondria-related processes. Our extensive in vitro validation experiments including extracellular flux assay and metabolome analysis confirmed that TRAF6 loss in AML cells induces dynamic changes in metabolites and the impairment of mitochondrial function. To identify the mechanism of metabolic alterations by TRAF6 loss in leukemia, we compared the upregulated genes in TRAF6-knockdown AML cells with the 130 previously identified essential genes for AML cell survival in vitro and in vivo ( Yamauchi et al. Cancer Cell). We focused on O-GlcNAc transferase ( OGT) as a gene associated in mitochondrial function and AML cell survival. Importantly, TRAF6 gene expression in human AML patient samples was positively correlated with the expression of OGT. To confirm the importance of OGT in the regulation of mitochondrial function mediated by TRAF6 in leukemia, we utilized the inhibitor of O-GlcNAcase (OGA), a remover of O-GlcNAc from its substrates. The treatment with MK8719 (OGA inhibitor) mildly, but significantly, restored the number of TRAF6 knockdown AML cells, which was correlated with the changes in mitochondrial function, indicating that O-GlcNAc modification regulated by TRAF6 is important for AML progression. In summary, We provide evidence for the oncogenic function of TRAF6 in leukemia, and shed light on the novel TRAF6/OGT/O-GlcNAc axis that regulates the metabolic reprogramming required for leukemogenesis.

Targeting ADSS2 Enhances BH3 Mimetics Treatment Induced Mitochondrial Apoptosis in AML Cells

Recently, the BH3 mimetic-Bcl2 inhibitor venetoclax (VEN), has been approved by FDA in 2018 for the treatment of patients with AML in combination with a hypomethylating agent (HMA) such as azacytidine (AZA). This synergy is likely due to HMA-induced mitochondrial vulnerability associated with cytoplasmic RNA metabolism. Nonetheless, most AML patients treated with the combination eventually relapse, possibly due to persistence of tumor cells not dependent on antiapoptotic proteins (Bcl2, Mcl1). There is a pressing need to develop targeted therapeutics sensitizing AML cells to mitochondrial apoptosis, particularly for those patients failed from VEN associated treatment. To achieve the goal, we performed a CRISPR/Cas9 Knockout (KO) screen (using Bim peptide-based dynamic ΔBH3 profiling as read-out) targeting genes (n=534) involved in cytosolic nucleotides metabolism pathways to increase mitochondrial vulnerability in a BH3 mimetic-refractory AML cell line (HEL). That search identified ADSS2, which catalyzes the first committed/rate-limiting step of AMP biosynthesis from IMP, emerged as the top hit, exhibiting highest ΔBH3 priming. Subsequent BH3 priming assays validated that ADSS2 KO significantly enhanced sensitivity to BH3 mimetics across various AML cell lines, including MOLM13, SKM1, OCI-AML3, and HEL cells. ADSS2 KO also led to a noteworthy reduction in the IC50 values of both VEN and the Mcl1 inhibitor S63845 in these cell lines. Additionally, the synergistic effect between sgRNA targeting ADSS2 and VEN or S63845 was also confirmed in Cas9 expressing cells from two AML patient-derived-xenograft (PDX) samples. We next transplanted AML cells (form one PDX samples) with or without ADSS2-KO into NGS mice, then treated mice with VEN. Notably, VEN treatment (100 mg/kg, QDx14, PO) significantly reduced tumor burden in ADSS2-KO AML mice compared to WT AML mice. Additionally, ADSS2 KO combined with VEN treatment group displayed a survival advantage compared to single VEN treatment group. Further mechanistic investigations revealed that ADSS2 KO led to a remarkable depletion of AMP rather than ATP, and a robust repression of AMPK activity in HEL cells. To assess whether AMPK inhibition mimics the effect of ADSS2 KO, AMPKα-deficient (Mx1-Cre/Prkaa1 fl/fl/ Prkaa2 fl/fl) MLL-AF9 (MA9) transformed murine AML cells were assessed. Indeed, AMPKα depletion substantially enhanced VEN treatment efficiency ex-vivo. Notably, enforced overexpression of a constitutively active AMPKa2 (T172D) construct in a VEN sensitive cell line SKM1, promoted cell resistance to mitochondrial apoptosis by VEN or S63845. Moreover, T172D expression can also abolish ADSS2-KO mediated mitochondrial vulnerability to VEN or S63845 treatment, indicating the pivotal role of AMPK signaling in mediating ADSS2 function. There are no reported ADSS2 inhibitors. To explore ADSS inhibitor, we conducted a fragment-based virtual screen of 8,000,000 compounds (MolPort) library and requested the top-ranking hits for ADSS2 catalysis evaluation by ELISA. Notably, the top two hits AS104 and AS71 exhibited the most potent inhibitory effects (AS104 IC50=2.43µM, AS71 IC50=3.85µM). NMR-STD analysis was conducted in a cell-free system, which unequivocally confirmed the physical binding of inhibitors and the ADSS2 protein. To further investigate the interactions between AS71 and AS104 with ADSS2 in a cellular context, both compounds were subjected to a cellular thermal shift assay (CETSA). Notably, treatment with AS71 and AS104 led to substantial shifts in ADSS2 protein thermal stability, providing evidence of their interactions with ADSS2 within the cellular environment. In both HEL cells and a primary VEN-resistant AML samples, treatment with AS71 and AS104 significantly increased ΔBH3 priming. Collectively, our observation revealed that targeting ADSS2 is critical for sensitizing AML cells to BH3 mimetics. We are now conducting preclinical assessments of ADSS2 inhibitors.

Dihydroartemisinin Synergistically Promotes the Antileukemic Activity of Venetoclax By Downregulating Mcl-1 in Acute Myeloid Leukemia Cells

Background: Venetoclax is a BH3 mimetic, which selectively inhibits Bcl-2. For older persons with newly diagnosed AML, the FDA has approved the use of venetoclax in conjunction with hypomethylating drugs or low-dose cytarabine. However, the selective inhibition of Bcl-2 in AML often results in the overexpression of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance. Dihydroartemisinin (DHA) is a herbal monomer that is taken orally to cure malaria. Studies have shown that DHA downregulates MCL-1 protein expression in AML cells. In the present study, we investigated whether the combination of venetoclax and DHA efficiently promotes apoptosis in AML cells. Methods: We assessed the combined effect of venetoclax and DHA on cell viability, apoptosis, combination index, and cell cycle in AML cells from AML cell lines and deno AML patients. Western blot analysis was used to validate the mechanisms of venetoclax and DHA. Results: Venetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with DHA; their synergism was aptly represented by the combination index. Venetoclax and DHA had significant synergistic inhibition of cell viability both in cells from AML cell lines and in deno AML patients. The combination of venetoclax and DHA impaired cell cycle progression by arresting the cell cycle in S-phase and inducing apoptosis. Mechanistically, DHA mitigated venetoclax-induced upregulation of Mcl-1 and BCL-XL. Moreover, the combination inhibited the expression of C-MYC. Conclusions: Our findings demonstrate the synergistic, preferential antileukemic effects of venetoclax and DHA on AML cells, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat AML.

NKX101, an Allogeneic Off-the-Shelf CAR NK Cell Therapy Targeting NKG2D-Ls, Has Potent Anti-Leukemic Activity Alone or in Combination with Ara-C

Introduction NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L + cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD -). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC 50 and IC 90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 cells and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells. Conclusions In summary, we show that NKX101 potently targets and eliminates AML cells expressing NKG2D-Ls further validating NKG2D-Ls as therapeutic targets. Additionally, we show that the combination of Ara-C and NKX101 leads to increased anti-leukemic activity, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

Gamma-Delta (γδ) CAR-T Cells Lacking the CD3z Signaling Domain Enhance Targeted Killing of Tumor Cells and Preserve Healthy Tissues

Introduction: Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies for patients who have limited treatment options. Successful advancement of CAR-T therapy to myeloid hematopoietic and solid malignancies, however, has been limited by the potential of life-threatening on-target, off-tumor toxicities against healthy tissues, the natural tumor heterogeneity and consequent selection pressure leading to relapse. Addressing these limitations to widen the therapeutic index is crucial as we seek safer and more effective CAR-T therapies. In this regard, γδ T cells as first responders of immunity hold promise as they can directly identify and eliminate malignant cells through the recognition of multiple tumor-associated stress antigens not commonly expressed on normal tissues. We sought to combine the tumor-sensing capabilities of γδ T cells and the localization enhancement of CAR-T by excluding the CD3ζ domain in a non-signaling CAR (nsCAR) to allow the targeted killing against tumors, while sparing healthy tissues. Methods: Standard and nsCAR constructs against CD19, CD33 and CD123 were cloned into separate second-generation lentiviral vectors. The CAR constructs contained either a scFv targeting the respective antigen (anti-CD19/anti-CD33) or an IL-3 zetakine (IL-3z) targeting the CD123 followed by a flag-tag, a hinge, a transmembrane domain and a costimulatory domain + CD3ζ. IL-15 co-expression was incorporated to enhance γδ T cell fitness and persistence. CAR activation was determined by Jurkat T cells co-cultured with target ALL or AML cell lines followed by lentivector transduction of activated and expanded Vδ2 + γδ T cells. Cellular cytotoxicity was assessed at multiple E:T ratios against respective leukemia cell lines and healthy peripheral blood cells expressing the target antigen. Results: We observed significant upregulation of CD69 surface expression indicating activation of CD3ζ+ CD19CAR/CD33CAR/IL-3z Jurkat cells (3x, 20x, and 2x respectively) following culture with Nalm6 (CD19) and KG-1 (CD33 &amp; IL-3z). CD3ζ- ns19CAR/ns33CAR/nsIL-3z Jurkat cells did not upregulate CD69 in parallel cultures. We also observed a time-dependent reduction of the CD3ζ+ CD33 CAR + population over 7 days (43%) in Jurkat cells following extended coculture with KG-1 cells, while the nsCAR + Jurkat population remained unchanged, indicating the nsCAR may mitigate activation induced cell death (AICD). Ex vivo activated γδ T cells from healthy donors (N=4), were transduced with nsCAR lentiviral vectors with high efficiency (up to 80%). The ns19CAR cells effectively killed CD19 + Nalm6 cells and demonstrated enhanced (&amp;gt;1.5x) cytotoxicity compared to untransduced γδ T cells (UTD). After 48hr co-culture (E:T=2:1), the ns19CAR cells killed 79.7%+6.6% Nalm6 cells compared to 46.1%+7.2%) with UTD. Minimal cytotoxicity was observed for ns19CAR and UTD γδ T cells against B cells from healthy donor PBMC 5.2%+6.6% for ns19CAR vs. to -4.6%+7.7% with UTD. No significant difference in cytotoxicity against the CD19- K562 cells between the UTD or ns19CAR cells (73.5% vs. 71.5%, E:T=2:1) suggesting the enhanced cytotoxicity is ns19CAR directed. Similarly, ns33CAR and nsIL-3z cells, although requiring higher E:T ratios for similar killing efficiency, continued to demonstrate enhanced (up to 2.0x) killing against AML cells (HL-60, KG-1 and MOLM13) and CML cells K-562 compared to UTD in cytotoxicity assay with 24hr co-culture. Meantime, minimal cytotoxicity (&amp;lt;10%) was observed for ns33CAR, nsIL-3z, or UTD γδ T cells against CD33+ cells isolated from healthy donor PBMC. Conclusions: Ex vivo activated nsCAR cells efficiently recognize and kill leukemia cell lines while sparing peripheral blood cells bearing the same target antigen. The nsCAR cells also show increased cytotoxicity against leukemias over unmodified activated γδ T cells suggesting improvement in tropism and/or binding efficiency. In summary, our findings showed that the combination of nsCAR on γδ T cells may increase the therapeutic index to allow expansion of CAR-T therapy to cancers with unacceptable target expression on critical healthy cell populations. Further optimization of the nsCAR constructs may potentially enhance both the efficacy and safety profile of the next generation adoptive cell therapies against wider selection of cancers.

Novel Agents with Efficacy Against Cellular Models of Familial Platelet Disorder with Myeloid Malignancy (FPD-MM) Associated with Germline Mutant RUNX1

RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. In addition to mutant (mt) RUNX1, FPD-MM cells commonly harbor co-mutations involving the second allele of RUNX1, or BCOR, PHF6, K-RAS, WT1 or TET2. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need to develop novel targeted therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, conducted with the RNA-Seq signature induced by lethal RUNX1 knockdown in AML cells with mutant (mt) RUNX1, was reported by us to reveal homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole) as the top expression mimickers (EMs). In present studies, we demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in six patient-derived (PD) samples of FPD-MM vs RUNX1-FPD or normal CD34+ progenitor cells. ATAC-Seq and H3K27Ac ChIP-Seq analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater ATAC-Seq peaks and chromatin accessibility in FPD-MM cells with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the population of HSCs but concomitant increase in the macrophage population in FPD-MM (Fig. 1A). This was associated with positive enrichment of the mRNA expression belonging to the gene-sets (HALLMARK) of apoptosis signaling and TP53 targets while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample from a patient with Runx1 K194N, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. CRISPR knockout (KO) RUNX1 led to growth inhibition, such that GMR-AML1 cells that retain Runx1 outcompeted Runx1 KO cells within 20 days. Treatment with HHT induced loss of viability in GMR-AML1 cells (LD50: 40 nM). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation, DNA POLR2B, RRM2, CCND2 and PTPN11. Treatment with MB also dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 330 nM) as well as inhibited cell cycle and protein translation (via OPP Assay). CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the tail-vein infused and engrafted, luciferized GMR-AML1 cell xenograft, compared to the vehicle control, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of the NSG mice (p &amp;lt; 0.05) (Fig. 1B). These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM, including the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are effective against cellular models of FPD-MM versus RUNX1-FPD.

Menin Inhibitor Induced Menin Protein Degradation Contributes to Menin Inhibitor Efficacy

The chromatin adaptor protein Menin ( MEN1) is necessary for sustained proliferation in acute leukemias driven either by Lysine Methyltransferase 2A (MLL1/ KMT2A) gene rearrangements. Small molecule Menin inhibitors (MIs) disrupt the MENIN-KMT2A protein interaction by competing with KMT2A for a binding pocket in Menin. This disruption leads to a downregulation of Menin-KMT2A transcriptional programs, induces leukemia differentiation and decreases leukemic proliferation. Multiple MIs have progressed to clinical trials with promising early-phase results. However, a subset of leukemias that initially responded developed mutations in the MEN1 gene that result in mutant Menin proteins with decreased affinity to MI binding and thus drug resistance. This prompts the possibility that redesigned MIs might prevent development of this type of resistance. One potential novel feature of MI re-design considerations includes targeting Menin protein stability. Menin protein degradation has been observed following treatment with several MIs though this mechanism and its clinical utility is not well understood. Here, we characterize the mechanism by which Menin protein is degraded upon MI treatment as well as characterize the clinical implications of MI treatment induced Menin protein degradation. Using protein degradation CRISPR-based screening technology we have identified HECT ubiquitin ligase UBR5 as a regulator of Menin protein stability. Furthermore, depletion of UBR5 partially rescues the anti-proliferative effects of MI treatments through blunting transcriptional inhibition of canonical Menin/KMT2A target genes such as MEIS1/HOXA. UBR5 colocalizes with Menin and KMT2A proteins on chromatin and contributes to transcriptional inhibition during MI treatments. We further characterize Menin degradation by using CRISPR base editor screens coupled to FACS in an endogenously edited MEN1-mScarlet human leukemia cell line to identify key Menin residues responsible for Menin degradation after MI treatment. We found and validated, using Menin overexpression systems, residues near the KMT2A interaction interface that prevented degradation and are outside of the MI binding pocket. Our data suggests that Menin degradation necessitates MI induced KMT2A displacement and that UBR5/KMT2A possibly compete for Menin association at a putative degron. In our recent study we identified mutations that induce resistance to Menin inhibitors via base-editor screens and these mutations were also identified in AMLs from patients (Perner et al. 2023). In that screen we also observed a mutation (D136N) that is outside the Menin MI-binding pocket and conferred a selective advantage on AML cells during MI treatment; this mutation has not been observed in patients but suggests a yet uncharacterized resistance mechanism. We generated MEN1-D136N mutated human leukemia cell lines and observed both protein degradation rescue as well as MI resistance, albeit less so than binding pocket mutations. Menin/KMT2A-FITC-peptide fluorescence polarization assays suggest that MI binding is not affected by D136N. This suggests an alternative resistance mechanism mediated by evasion of Menin degradation. We next applied our base editing technology in MEN1-M327I mutated human leukemia cell lines to ask if degradation evasive compound mutations could deepen resistance phenotypes. We observed that the D136N substitution was the top compound mutation hit conferring a selective advantage in M327I MEN1 cells during a MI treatment course in our screen. Finally, we compared the Menin degradation potential of several MIs currently under clinical investigation and discovered variability among MIs for induced Menin degradation suggesting this is a designable feature. Additionally, we found that Menin degradation is not tightly linked to drug binding affinities opening the potential to overcome MI drug binding mutations through degradation mechanisms. Taken together our data suggests that MI induce degradation is an essential and designable feature of MI design/efficacy. Our data also suggests that degradation evasive mutations in MEN1 could be an alternative resistance mechanism to contend with going forward in clinical trials.

Unveiling Putative Pathway Enrichment Following Crisantaspase Treatment in AML through RNA Sequencing Analysis

Background: Tumor development is intricately influenced by metabolic reprogramming, resulting in the emergence of exploitable vulnerabilities that can be targeted. Glutamine (Gln) depletion has emerged as a promising therapeutic strategy for malignancies, including AML, that heavily rely on Gln for their survival. Erwinia-derived asparaginase, also called crisantaspase, has exhibited anti-AML activity in both preclinical and clinical models by disrupting Gln supply, albeit with the persistence of residual leukemia burden. To identify cellular pathways modulated after crisantaspase-induced Gln depletion, here we report our comprehensive results using a whole transcriptome profiling approach (RNA-Seq) after crisantaspase treatment. Methods: Whole-transcriptomic profiling using RNA-Seq was performed to profile gene expression of MOLM-14 AML cells following crisantaspase treatment. RNA was isolated from each sample and total RNA was utilized for this study. The samples were sequenced at IGS using the Illumina HiSeq sequencing platform. Raw sequencing reads generated for each sample were analyzed using the CAVERN analysis pipeline. Read quality was assessed using the FastQC toolkit to ensure good-quality reads for downstream analyses. P values were generated using a Wald test implemented in DESeq2 and then corrected for multiple hypothesis testing using the Benjamini-Hochberg correction method. Results: We utilized Rylaze, an FDA-approved Erwinia crisantaspase, to deplete exogenous Gln. MOLM-14 cells were treated with Rylaze for 16h, followed by whole-transcriptome/gene expression profiling (GEP) using RNA-seq. The transcriptomic analysis provided information on the regulation of gene expression between vehicle control and Rylaze treatment in AML, as well as on the potential association with several putative pathways including serine family amino acids biosynthetic process (GO: 0009070, p-value = 2.18E-08), regulation of programmed cell death (GO:0043067, p-value = 7.96E-06), and amino acid transmembrane transport (GO:0003333, p-value = 5.10E-05). Furthermore, Figure 1 illustrates the gene ontology terms enriched by the DEGs, offering insights into the underlying molecular mechanisms involved in the response to Rylaze-induced Gln depletion in MOLM-14 cells. For example, the upregulation of genes associated with the serine family amino acid biosynthetic process may suggest a compensatory mechanism to counteract the depletion of Gln induced by Rylaze. By employing generalized linear regression models, we observed significant changes in gene expression following Rylaze (141 genes) compared to the vehicle control. This finding is further illustrated by a heatmap generated from transcriptome data analysis (Figure 2). We identified 141 significantly differentially expressed genes (DEGs); among the Rylaze-treated samples, consisting of 114 up-regulated and 27 down-regulated genes. Specifically, we focused on five genes that exhibited significant modulation in response to the treatment with Rylaze. These genes included CHAC1, ADM2, S100P, CTH and SPOCK2. These genes might play crucial roles in the cellular response to Gln depletion and could potentially serve as biomarkers or therapeutic targets in AML treatment. Validation of these observations was performed using immunoblotting techniques and quantitative real-time polymerase chain reaction (qRT-PCR), which confirmed the significant increase in CHAC1, ADM2, S100P, and CTH, as well as the significant decrease in SPOCK2 (p&amp;lt;0.0001) following Rylaze-induced Gln depletion. These findings contribute to a better understanding of the drug's mode of action and the impact of Gln depletion on cellular processes in AML. Conclusion: This study represents a significant contribution towards advancing our understanding of amino acid-regulated metabolic pathways in AML, with potential implications for other Gln metabolism-dependent cancers as well.

DNA Methylation Instability: A Novel Biomarker for Aging, Clonality, and Cancer

Background: Clonal hematopoiesis (CH) is an age-related condition defined by the over-representation of blood cells derived from a single clone. CH has been associated with an increased risk of leukemia development, adverse cardiovascular events, and all-cause mortality. There has been great emphasis on investigating CH at the genomic level, yet the epigenetic factors that regulate the development and progression of pre-malignant clones haven't been extensively studied. Methods: Here, we develop a novel framework that employs DNA methylation (DNA-M) to detect abnormal clonal expansion of hematopoietic cells. DNA-M profiles of &amp;gt;1500 blood samples from healthy, young persons (age = 18) were interrogated to identify CpG sites with highly consistent methylation levels across individuals. DNA methylation instability (DMI), which we define as deviation in methylation levels at the identified stable methylation sites (SMSs), was evaluated in diverse healthy and cancer cohorts to investigate how epigenetic mechanisms may affect cellular functions resulting in clonality. Results: Characterization of SMSs. SMSs were found to be predominantly unmethylated and significantly enriched within CpG islands (OR: 13.8, p &amp;lt; 2 x 10 -16) and gene promoter regions (OR: 4.8, p &amp;lt; 2 x 10 -16). A gene ontology enrichment analysis implicated the corresponding genes in vital cellular processes such as DNA repair, damage response, and cell cycle checkpoints (p &amp;lt; 0.002). Consistently low methylation levels at SMSs were observed across major blood cell types. Moreover, analysis of a methylome cell atlas derived from whole genome bisulphite sequencing (WGBS) of 40 healthy human tissues revealed that SMSs remain stably unmethylated in the vast majority of tissues, suggesting broad and strict regulation at those sites. DMI in overt blood cancers. Interrogation of SMS methylation levels across diverse control and cancer datasets revealed significantly higher destabilization in cancer samples (Fig. 1, p &amp;lt; 2 x 10 -16). Moreover, longitudinal profiling of bone marrow samples from AML patients (n = 4, four time points each) indicated that DMI levels follow the clonal burden patterns expected during treatment. Analysis of serial dilutions of AML cell lines in cord blood further corroborated the link between DMI levels and malignant cell fractions (p &amp;lt; 2 x 10 -16). DMI in non-cancer cohorts. Given the link between DMI and clonality, we measured DMI in blood samples of three independent aging cohorts of individuals without a malignancy (n = 1751, age 14-101) and observed a positive correlation with age (r = 0.25, 0.38, 0.4; p &amp;lt; 7 x 10 -11). These results are in accordance with the increasing incidence of CH with age and suggest that DMI, much like genomic mutations, may causally precede the development and progression of various age-related diseases. CH has previously been associated with adverse cardiovascular outcomes. In a competing risk analysis of 64 cardiogenic shock patients (CH+ = 31, CH- = 33), DMI was better able to stratify patients into risk groups than genomics-based CH detection (Kaplan-Meier analysis, CH: p = 0.46, DMI: p = 0.01). These results indicate that DMI is significantly associated with mortality in high-risk patients. DMI and gene regulation. WGBS of AML and healthy control samples revealed that significantly destabilized SMSs in AML (p &amp;lt; 7 x 10 -6) near transcriptional start sites reflect broader hypermethylation of promoter regions. Transcriptome profiling of age-stratified controls (n = 755) and AML samples (n = 151) confirmed a gradual decrease in expression for many of the implicated genes. Notably, genes known to suppress cell proliferation and promote apoptosis such as BASP1 were implicated (Fig. 2, r = -0.20, p &amp;lt; 2 x 10 -16), as well as novel genes that have not previously been associated with age-related gene silencing and leukemia development. These findings further emphasize that DMI reflects a gradual process that precedes disease. Conclusion: Our results suggest that the development of clonality is not merely a genomic phenomenon and that the gradual silencing of specific genes via DMI in their promoter regions may result in positive selection and clonal dominance. This work has significant implications for elucidating the molecular mechanisms underlying age-related diseases, early detection of cancer, and the development of novel biomarkers and therapeutic strategies against hematologic malignancies.

Anti-Leukemic Activity of Luveltamab Tazevibulin (LT, STRO-002), a Novel Folate Receptor-α (FR-α)-Targeting Antibody Drug Conjugate (ADC) in Relapsed/Refractory CBF2AT3::GLIS2 AML

Introduction: CBFA2T3::GLIS2 (CBF-GLIS) is an oncogenic fusion associated with a lethal and highly refractory childhood myeloid leukemia manifested by the RAM phenotype, uniquely expressed in infants and young children. Conventional chemotherapy and stem cell transplant (SCT) result in extraordinarily high rates of induction failure (&amp;gt;80%) and near uniform fatality with 2-year survival &amp;lt;15%. When conventional therapy fails, this leukemia is refractory to all reinduction therapies with post-relapse survival of nearly 0%. We have previously reported that FR-α is expressed on the surface of CBF-GLIS AML cells and that LT, an investigational ADC, demonstrated potent in vitro and in vivo anti-leukemia activity in CBF-GLIS AML cell lines and xenograft models (PMID: 36149945). LT has been provided under single patient IND to pts with CBF-GLIS AML. Herein, we update the clinical experience. Methods: All pts were required to demonstrate CBF-GLIS fusion by PCR or sequencing. Each investigator in consultation with Sutro Biopharma Inc. defined the treatment plan as to dose, schedule, and combination therapy administered. Results: Between August 2021 and March 2023, 25 pts with relapsed/refractory CBF-GLIS AML were treated with LT at doses of 4.3 or 5.2 mg/kg every 2-4 weeks (4 received smaller fractionated doses (FD) on days 1, 3, 5 per cycle). Median age at treatment was 2 years (range 0.7-8) with median number of prior therapies of 2 (range 1-7). 10 pts had relapsed after SCT. The remaining 15 had primary refractory, relapsed or MRD+ AML. Median time from initial diagnosis was 7.9 months (range 1.5-82.5). Of the 25 pts who received LT, 18 had ≥5% blasts (morphologic disease, MD) and 7 had &amp;lt;5% blasts (sub-morphologic disease, SMD). 21 pts initially received LT monotherapy (mono) (15 with MD and 6 with SMD), 9 of which went on to receive combination (combo) with additional therapies. The remaining 4 received LT in combo upfront. Morphologic or molecular/MRD- response was observed in 12/25 pts (48%). Among those with MD, 7/18 (39%) reached a CR/CRh (5 MRD-) and among those with SMD, 5/7 (71%) reached MRD- CR. Median number of doses received before response was 2. Responses were observed among patients receiving both mono and combo. Among the 11 MD pts treated with mono (excluding FD), 3 reached CR (27%). Among 10 pts with MD who received combo either initially or following mono, 4 reached CR/CRh (40%). Among the SMD pts, 4 reached MRD- with monotherapy and 1 with combo. Of the 10 pts with post-SCT MD relapse, 7 patients received non-fractionated LT either as mono or combo therapy. Of these, the CR rate was 71% (5/7). LT therapy enabled these children to receive potentially definitive therapy. Of the 7 pts with MD in CR, 4 proceeded to SCT, 2 received donor lymphocyte infusions and 1 received consolidation therapy. Of the 5 pts with SMD in MRD- CR, 1 proceeded to SCT, 2 received consolidation therapy in preparation for SCT, and 2 received LT maintenance therapy. In total, 12 pts remain on treatment. Of the 25 pts who received LT, 17 (63%) are alive, 7 died of progressive disease (5 with relapse post-SCT) and 1 died from SCT-related complications. Of the 10 pts who received LT (non-fractionated) post-transplant, 7 are alive (78%). Pt status and treatment are shown in Figure 1. LT was generally well tolerated. TEAEs reported at an incidence of ≥ 25% included decreased neutrophil, platelet, WBC, or lymphocyte count, anemia, increased AST or ALT, diarrhea, and pyrexia. Grade 3+ TEAEs (≥ 10%) included decreased neutrophil, platelet, WBC, or lymphocyte count, anemia, febrile neutropenia, ALT increased, vomiting and hypoxia. Observed cytopenias were often reported in pts with baseline cytopenia. Grade 5 SAEs were limited to fractionated dosing pts: apnea (n=1) and cardiopulmonary failure (n=1). Neither event was considered related to LT. The median duration of LT treatment was 9.1 weeks (2.1-60) and 60% of pts received ≥5 doses. While the initial dose was typically delivered inpatient, approximately 80% of subsequent doses were delivered outpatient. Conclusion: LT based therapy in CBF-GLIS AML provides clinical and laboratory responses that are highly unusual in this refractory patient population. LT has an acceptable safety profile and does not require inpatient delivery. Importantly, LT appears to be enabling these children to receive potential definitive therapy. These data support further characterization of this drug in pediatric CBF/GLIS AML.

Multimodal Atlas of Paired Diagnosis and Relapse AML Samples Enables Novel Therapeutic Targeting of Surface Antigens

Note: A.H. and M.U. share co-first authorship. AML is an aggressive clonal malignancy characterized by combinations of chromosomal abnormalities, gene mutations, and cell surface antigen (Ag) expression profiles. This heterogeneity contributes to refractory or relapsed disease that presents a major challenge when treating AML. Previous single cell sequencing studies of primary AML samples have provided remarkable insight into the clonal architecture of AML cell populations and how their mutational, transcriptomic, and surface Ag profiles vary between patients. However, information addressing clonal shifts during progression from diagnosis to relapse in large cohorts is lacking. Here, we adapted single cell RNA sequencing with surface Ag feature barcoding to analyze more than 450,000 cells from 28 paired AML patient bone marrow mononuclear cell samples collected at diagnosis and relapse (56 samples). To our knowledge, it is the largest and most comprehensive single cell AML atlas to date. This atlas contains rich clinical metadata including cytogenetics, mutation status of canonical AML genes, treatment history, and survival information. We leveraged these data to identify potential correlations with clonal heterogeneity during progression to relapse and to propose novel strategies for targeting the surface of AML cells. The feature barcoding panel consists of a comprehensive list of 81 surface Ags reported in clinicaltrials.gov or mined from literature, including large proteomics mass spectrometry datasets of AML patient bone marrow samples (deBoer et. al. Cancer Cell 2018; Jayavelu et al, Cancer Cell 2022). Feature barcoding produces antibody derived tag (ADT) counts which are interpreted as relative values but alone does not indicate absolute number of Ags per cell. We addressed this limitation by measuring absolute Ag density of AML Ags, CD33, CLL1, CD123, and EMR2 in patient samples using the flow cytometric QuantiBRITE assay. Focusing on these four Ags, we modeled the relationship between ADT expression and antibodies per cell (ABC) Ag density from QuantiBRITE. The model was applied to impute absolute Ag density for the remaining 77 Ags in all samples. UMAP visualization after sample transcriptome integration revealed distinct clustering of CD45-dim myeloblasts, T cells, B cells, and erythroid cells. Longitudinal analysis identified 28 surface Ags that were differentially expressed (absolute log 2FC &amp;gt; 1, p &amp;lt; 0.01) between relapse and diagnosis myeloblasts across multiple patients (Fig. 1A). An additional 19 Ags were differentially expressed in no more than one patient while the other 19 had no significant change in any patient, highlighting the inter-and intra- heterogeneity of AML at diagnosis and relapse in these patients. Targeting multiple antigens simultaneously may help address the issue of antigen heterogeneity of tumor cells and help avoid potential antigen escape. Because multi-targeting therapies are a potential solution to this, weleveraged the single-cell resolution of our atlas to systematically identify Ags that in combination provide maximum coverage and are significantly co-expressed in myeloblasts. From this analysis, we found 48 and 52 co-expressed Ag pairs in diagnosis and relapse blasts, respectively (r &amp;gt; 0.4, p &amp;lt; 0.01; Fig. 1B). Of note, CD44 and EMR2 were co-expressed at both diagnosis (r = 0.49 p &amp;lt; 0.01) and relapse (r = 0.3, p &amp;lt; 0.01) with &amp;gt;90% of their blasts expressing CD44, EMR2, or both at targetable antigen levels of &amp;gt;1000 Ags/cell. Furthermore, 10 samples had &amp;gt;40% of their blasts co-expressing both Ags simultaneously at &amp;gt;1000 Ags/cell at diagnosis or relapse. 6 of these patients had a &amp;gt;10% increase in co-expression at relapse, 3 had &amp;gt;10% decrease at relapse, and one patient had no co-expression change. Our comprehensive profiling of AML has provided high resolution insight on cell surface at diagnosis and during disease progression and produced novel hypotheses about targetable combinations of surface Ags based on their expression changes between timepoints, co-expression, and surface densities. Furthermore, our reference atlas can be further dissected to characterize the transcriptome and mutational profiles of myeloblasts at diagnosis and relapse thereby prodding an important resource to create surface antigen protein - transcript correlations within AML blasts and to trace molecular signatures during disease progression.