Acute myeloid leukemia (AML) is a hematologic malignancy with high relapse rates and limited treatment options due to extensive intra-tumor heterogeneity across patients. To characterize this heterogeneity, we profiled matched bone marrow mononuclear cell (BMMC) samples from 26 patients with adult AML at diagnosis and relapse using the cellular indexing of transcriptome and epitope sequencing (CITE-seq) and quantitative flow cytometry. These data together represent a comprehensive multimodal and longitudinal single-cell resource that reveals the transcriptomic and immunophenotypic landscape of AML. Data integration of CITE-seq and flow cytometry surface antigen readouts enabled systematic quantitation of surface antigen co-expression across individual leukemic cells, providing a granular framework for the design of immunotherapeutic strategies to target heterogeneous AML. With this resource, we identified CD33, CLL-1, LAIR1, ITGA4, DEC-205, and CD244 as antigens that induced cytotoxicity in AML cell lines in vitro when co-targeted by antibody drug conjugates (ADCs) or chimeric antigen receptor T (CAR-T) cells, demonstrating the exploitation of AML heterogeneity for immunotherapeutic innovation.

VCAR33, a donor-derived CD33-directed chimeric antigen receptor (CAR) T cell product, was developed to decrease relapse of high-risk acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation (alloHCT). We describe pre-clinical characterization of the VCAR33 construct, which was optimized for long-term anti-tumor surveillance based on killing and persistence assays. Prior to its use in post-alloHCT maintenance, we evaluated safety and efficacy of VCAR33 in a phase 1/2 clinical study for adults with relapsed or measurable residual disease (MRD)-positive CD33+ AML/MDS after alloHCT. Fifteen patients received VCAR33 across 2 arms stratified by disease burden: 7 patients in Arm A (bone marrow blasts ≥ 5%) at dose level 1 (DL1; 1 x 106 CAR+ T cells/kg) and 8 patients in Arm B (bone marrow blasts < 5%) at DL1 (n=5) and DL2 (3 x 106 CAR+ T cells/kg; n=3). The study ended for non-safety reasons before escalation to DL3 (1 x 107 CAR+ T cells/kg) and maximum tolerated dose was not determined. The most common treatment-related adverse event was cytokine release syndrome (93.3%; all < grade 3). Four patients (26.7%) experienced immune cell-associated neurotoxicity syndrome (1 ≥ grade 3) and 1 patient (6.7%) had grade III acute graft-versus-host disease within 28 days of VCAR33 infusion. Fourteen patients (93.3%) had transient VCAR33 expansion. Overall response rate was 20%: 2 patients had complete remission with incomplete count recovery in Arm A and 1 Arm B patient achieved MRD clearance. This allogeneic CAR T product demonstrated acceptable safety and preliminary anti-leukemic activity. ClinicalTrials.gov: NCT05984199.

Developing novel therapeutics requires robust early-stage target de-risking to ensure safety and efficacy. We developed a scalable proteogenomic framework integrating population-scale human genetics and plasma proteomics to identify genes tolerant of inactivation (i.e., dispensable) within hematopoietic compartments, thereby enabling safer targeted immunotherapies. Using CD33 as a validated benchmark, we observed that naturally occurring loss-of-function (LoF) variants lead to concordant RNA and protein depletion, supporting functional gene inactivation. Early clinical results from the Trem-Cel trial ( NCT05945849 ) further provide proof of concept that deletion of dispensable antigens can enable safe and effective immunotherapy in humans. We extended this approach genome-wide in the UK Biobank and identified 237 candidate dispensable genes, filtered by plasma proteomic data and hematopoietic expression, highlighting LY75 (CD205) as a novel candidate with strong proteogenomic evidence of LoF tolerance. This work establishes a generalizable, quantitative proteogenomic framework for systematic prioritization of dispensable gene targets for editing, providing a foundation for next-generation cell and gene therapies that minimize on-target, off-tumor toxicities.

Acute myeloid leukemia (AML) remains a major therapeutic challenge due to extensive disease heterogeneity and lack of cancer-specific antigens. ADGRE2 has emerged as a promising AML target with broad expression in AML patient blast and leukemic stem cell-enriched populations. However, comparable expression in healthy hematopoietic stem and progenitor cells (HSPCs) and myeloid lineages suggests a high susceptibility to on-target, off-tumor myelotoxicity with ADGRE2-targeted therapies. Guided by human genetics data identifying loss-of-function variants, we evaluated whether ADGRE2 is dispensable in hematopoietic stem cells as a protective approach for transplant-based shielding from ADGRE2-directed therapies. Using CRISPR-Cas9 and adenine base editors, we achieved high-efficiency ADGRE2 knockout (>94%) in HSPCs with corresponding protein loss without impairing cell viability, differentiation, and cytokine release in vitro , or long-term engraftment, multilineage differentiation, and persistence of gene editing in mouse xenografts. We also developed novel ADGRE2-specific chimeric antigen receptor (CAR) T cells that demonstrated potent cytotoxicity against AML cells, even at low antigen levels. Together, these findings establish ADGRE2 as a compelling AML target and provide a framework for hematopoietic stem cell transplant with protective gene editing to enable ADGRE2-directed immunotherapies while minimizing myelotoxicity. ### Competing Interest Statement Y.K, J.E., M.S., J.X.F., N.M., A.H., H.Q., R.W., M.I.L., H.G.G., J.S., T.C., and J.R.L. are salaried employees of Vor Biopharma and may hold equity in the company. J.E., M.S., J.X.F., A.H., R.W., M.L., H.G.G., J.S., T.C., and J.R.L. are inventors on patent applications assigned to Vor Biopharma Inc. Vor Bio, Cambridge, US

Note: A.H. and M.U. share co-first authorship. AML is an aggressive hematologic malignancy with few treatment options beyond chemotherapy and hematopoietic cell transplantation (HCT). Previously, we introduced a multimodal AML atlas of paired patient primary bone marrow mononuclear cell (BMMC) samples collected at diagnosis and relapse from a large patient cohort, revealing extensive inter- and intra-patient blast antigen (Ag) heterogeneity. We have since expanded this atlas to include additional samples from healthy donors, allowing us to categorize blasts into hematopoietic cell lineages. We leverage this large dataset of blast heterogeneity across 68 samples to identify novel Ags that can be targeted with multi-specific immunotherapies. In addition, we determined the number of target Ag molecules presented on individual blasts (Ag count), since immunotherapies including CAR-T cells and antibody drug conjugates (ADC) show varying sensitivities to Ag counts for effective blast recognition and killing. Here, we identify Ag combinations at therapeutic thresholds amenable to multi-specific immunotherapy and validate their targetability with in vitro cytotoxicity assays using CAR-T cells and ADCs. Nearest-neighbor projection of AML BMMCs to the annotated healthy donor reference revealed considerable heterogeneity in blast state composition across both patients and timepoints suggesting that multi-specific targeting can enable comprehensive targeting of blast populations. As described previously, all samples in our atlas include both feature barcoding with antigen derived tags (ADT) on 81 Ags and QuantiBRITE flow cytometric data, measured as antibodies bound per cell (ABC), on four well-characterized AML Ags: CD33, CLL-1, CD123, and ADGRE2. Using patient-matched data from these two assays, we trained random forests to model the relationship between ABC and ADT readouts for these Ags and applied it to estimate Ag count for the remaining 77 Ags on individual blasts for all samples. This represents a novel method for integrating sequencing and fluorescence data with machine learning to infer Ag counts. Effective target Ag combinations are collectively expressed across blasts and co-expressed on individual blasts to enable recognition by multi-specific therapies and guard against Ag escape. To demonstrate this on established AML Ags, we developed novel multi-specific CAR-T cells with an “OR” logic gate directed against CD33 and CLL-1. The lead candidates significantly reduced tumor growth, induced T cell expansion, extended animal survival, presented greater persistence in hematological compartments, and showed osteotropic activity in HL-60-based in vivo models of AML. As observed in our atlas, CD33 and CLL-1 cover a considerable fraction of blasts but 25% of relapse samples had <80% of blasts expressing either CD33 or CLL-1 at a threshold of 1000 Ags/cell, a sufficient therapeutic threshold for some immunotherapies. Thus, to increase the likelihood of targeting all blasts we ranked Ags in our atlas which, when combined together, or with CD33 and CLL-1 label the maximum number of blasts in the most patients. We identified four additional Ag candidates: LAIR1, ITGA4, LY75, and CD244 that expressed >1000 Ags/cell on >80% of cells in 22, 24, 14, and 7 of the 28 diagnosis samples, and 21, 25, 14, and 10 of the 28 relapse samples, respectively. As validation, we used QuantiBRITE to measure ABCs of these four Ags on four samples selected from our atlas repository. We observed high consistency between the measured ABC readout and the predicted ABC for all four Ags and samples (Pearson r = 0.87, p = 9.9e-6). Next, we evaluated therapeutic targeting of these Ags individually using in vitro ADC cytotoxicity assays with MOLM-13 cells, which provides a sensitivity measure of the ADC as a function of Ag expression. We observed >50% ADC-mediated cell killing at <0.1nM of primary antibody for all four Ags, supporting their therapeutic targetability. In summary, we integrated rich multimodal data from our atlas with machine learning and performed experimental validation to identify promising single and combinations of AML Ag targets including CD33, CLL-1, LAIR1, ITGA4, LY75, and CD244. These novel combinations can be leveraged to develop new multi-specific agents (e.g. CAR-T cells or ADCs) that better address AML heterogeneity.

The underlying gene regulatory networks (GRN) that govern leukemia stem cells (LSC) in acute myeloid leukemia (AML) and hematopoietic stem cells (HSC) are not well understood. Here, we identified GRNs by integrating gene expression (GE) and chromatin accessibility data derived from functionally defined cell populations enriched for HSC and LSC. We analyzed n=32 LSC+ and n=32 LSC- cell fractions from n=22 AML patients, along with n=7 stem and n=10 progenitor enriched cell populations sorted from human umbilical cord blood (hUCB), producing a database of n≈17,000 transcription factor (TF) regulatory interactions for hUCB-HSPC and AML. We developed an iterative algorithm that associates the degree of chromatin openness with TF binding preferences, and the GE of candidate TF and target genes within 100kb upstream of transcription start sites. A putative regulatory structure was found to be enriched in HSC-enriched cell populations, comprising TF-target gene interactions between ETS1, EGR1, RUNX2, and ZNF683 oriented in a self-reinforcing configuration. A regulatory loop comprising FOXK1 and MEIS1, rather than the 4-factor HSC subnetwork, was detected in the LSC-specific GRN. The core HSC and LSC TF networks were extended using protein-protein interaction (PPI) data to determine connectivity with interacting genes whose expression strongly associated with LSC/HSC frequency estimates, producing a database of n=103,516 PPI target pathways. The effect of perturbing genes along the identified pathways on functional HSC and LSC frequency was predicted based on statistical regression analyses. To validate GRN predictions, we used pharmacologic and CRISPR targeting, in addition to re-examining published functional data associated with several network nodes that were predicted to impact stemness. Notably, we found that inhibition of CDK6 in AML samples markedly reduced LSC numbers as assessed in de novo serial xenotransplantation studies (fold change ≈ 10), as predicted by the LSC GRN model. Additionally, in-house CRISPR-based knockdown of ETS1 resulted in a significant decrease in HSC quiescence-associated microRNA-126 expression, and increased HSC frequency. Taken together, our models provide a comprehensive view of the underlying regulatory structures governing functional human HSC and LSC. This approach has translational potential as it can be used as a high-throughput in-silico screening tool for the systematic identification of gene targets for LSC elimination and HSC expansion. ### Competing Interest Statement The authors have declared no competing interest.

Background Acute myeloid leukemia (AML) is the most common form of leukemia in adults. However, the clinical outcome for high-risk patients remains poor, highlighting the urgent need for the development of new therapeutic strategies [1]. Chimeric antigen receptor (CAR) T cell therapy holds promise as an immunotherapeutic strategy and targeting C-type lectin-like molecule-1 (CLL-1, CD371) represents an attractive approach, as CLL-1 is highly expressed on AML blasts and leukemic stem cells [1]. Here, we present preclinical data detailing the development and functional characterization of novel CLL-1-directed CAR-T cells to identify top candidates from a panel of 24 CLL-1 binders. Methods CLL-1-directed binders were identified by phage display technology and evaluated by flow cytometric, ELISA and Octet analyses. Selected binders were used to generate second generation CLL-1-directed CAR constructs with a 4–1BB co-stimulatory domain. CAR constructs were transduced into primary T cells using lentiviral vectors and investigated for antigen-specific cytolysis by flow cytometry-based assays. Co-culture experiments were conducted for 24 and 48 hours with CLL-1-expressing WT and CLL-1 knockout (KO) HL60 target cells. CAR candidates showing robust antigen-dependent activity were further evaluated for potency by bioluminescent-based assays at low effector to target (E:T) ratios, for long-term persistence in repeated stimulation assays, and for avidity measurements in acoustic force microscopy assays. The top CLL-1 CAR candidates were further studied in an in vivo murine xenograft model using HL60 AML cells in NSG mice. Results We completed an in-depth in vitro characterization of 24 CLL-1-directed CAR-T cells and identified top candidates that exhibited 1) potent and specific cytotoxicity of CLL1-expressing targets with minimal nonspecific killing of CLL1 KO targets, 2) high levels of antigen-dependent activation, and 3) significant antigen-dependent cytokine secretion. Importantly, top candidates effectively killed CLL1-expressing targets at low E:T ratios, demonstrated superior persistence after repeated stimulation with target cells, and displayed similar high binding avidity. Lead CLL-1 CAR-T cell candidates significantly reduced in vivo tumor growth as assessed by IVIS imaging and flow cytometric analyses, which also indicated tumor cell clearance and CAR T cell expansion. Conclusions Altogether, our preclinical data demonstrate highly efficacious and antigen-specific CLL-1-directed CAR-T cells, with potent in vitro and in vivo cytolytic activity. These results support further clinical development of the lead CLL-1 CAR candidate either as a stand-alone treatment or in combination with the VOR eHSC platform to eliminate on-target off-tumor toxicity to fully benefit high-risk AML patients in need.

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 > 1, p < 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 > 0.4, p < 0.01; Fig. 1B). Of note, CD44 and EMR2 were co-expressed at both diagnosis (r = 0.49 p < 0.01) and relapse (r = 0.3, p < 0.01) with >90% of their blasts expressing CD44, EMR2, or both at targetable antigen levels of >1000 Ags/cell. Furthermore, 10 samples had >40% of their blasts co-expressing both Ags simultaneously at >1000 Ags/cell at diagnosis or relapse. 6 of these patients had a >10% increase in co-expression at relapse, 3 had >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.

INTRODUCTION: Acute myeloid leukemia (AML) is the most common form of adult acute leukemia. Currently, allogeneic hematopoietic stem cell transplant (HCT) is the standard of care for high-risk patients yet ~40% patients will relapse, emphasizing the need for new therapeutic approaches. Effective targeted therapy in AML has been hampered by a lack of cancer specific targets. One described AML target is CD33, a glycoprotein expressed predominantly on normal myeloid cells and the majority of AML cells. However, CD33-directed therapies have been challenged by cytopenias due to on-target, off-tumor toxicity and, in the case of bispecific antibodies, cytokine release syndrome (CRS). Tremtelectogene empogeditemcel (trem-cel) is a gene-edited hematopoietic stem and progenitor cell (HSPC) transplant product currently in clinical trial (NCT04849910) whereby CD33, shown to be dispensable for normal hematopoiesis and function, is deleted to allow for post-HCT treatment with CD33-directed immunotherapies with reduced myelotoxicity as the reconstituted hematopoietic compartment lacks CD33. JNJ-67571244, a bispecific antibody that binds to both CD3 and CD33, is capable of inducing T cell recruitment for CD33-directedtumor cytotoxicity. This study examines whether CD33 neg hematopoietic cells are protected from JNJ-67571244 and if CD33 neg human HSPC (hHSPC) xenotransplanted mice have reduced levels of inflammatory cytokines associated with CRS when treated with JNJ-67571244. METHODS: In vitro proof-of-concept (POC) studies were carried out using CD3 + T cells co-cultured with JNJ-67571244 and AML target cell lines. Further studies were conducted with CD34 + hHSPCs that were either CRISPR/Cas9 gene edited to delete CD33 or treated with a non-targeting gRNA control (gCtrl) followed by differentiation towards the monocytic lineage. These in vitro differentiated monocytes (MIVD) were assayed for editing frequency, CD33protein expression and myeloid phenotype by flow cytometry prior to co-culture with donor matched T cells and JNJ-67571244 ranging from 0.5 pM to 500 nM. Cytotoxicity was analyzed using flow cytometry at 48 and 72 hr and cytokine concentrations in the supernatants were measured using Luminex. For in vivo POC studies, CD33 or gCtrl-edited CD34 + hHSPCs were xenotransplanted into NSG-SGM3 mice followed by treatment with either control bispecific (CD3xNull) or JNJ-67571244 at 0.5 mg/kg. Plasma cytokine concentration were measured via Luminex and hematopoietic tissues were analyzed by flow cytometry to measure protection from JNJ-67571244 cytotoxic killing. RESULTS: Annexin V and Live/Dead staining showed that JNJ-67571244 was able to induce AML target cell killing by human T cells. Importantly CD33 neg MIVD cells co-cultured with T cells and JNJ-67571244 were significantly more viable at all doses compared to gCtrl cells. The EC 50 of gCtrl MIVD was 0.0208 nM whereas the CD33 neg MIVD EC 50 was at least 20,000-fold higher (>430.8 nM). T cell activation, measured by CD25 and CD69 positivity, and release of cytokines that are associated with CRS such as IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in the CD33 neg MIVD cells treated with JNJ-67571244 compared to the gCtrl group at all doses. Analysis of blood and bone marrow from xenotransplant studies revealed that JNJ-67571244 was effective at killing CD33 + cells as well as CD14 + monocytes in the gCtrl group treated with JNJ-67571244. Conversely, CD14 + monocytes and CD15 + neutrophils derived from CD33-edited hHSPCs, were not significantly reduced in cell number when treated with JNJ-67571244 suggesting that they were protected from JNJ-67571244 specific killing. IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in mice transplanted with CD33-edited hHSPCs compared to gCtrl group in response to JNJ-67571244. CONCLUSIONS: Taken together, these studies demonstrate that CD33 deleted hematopoietic compartment is protected from the CD33 directed immuno-therapy JNJ-67571244 both in in vitro cytotoxicity assays and preclinical xenotransplantation studies, with decreased concentrations of inflammatory cytokines associated with CRS. These findings enable the development of a next-generation AML treatment strategy by pairing trem-cel transplant with a subsequent CD33-directed bispecific compound to potentially improve safety and efficacy while minimizing myelotoxicity.

Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Here, we describe preclinical studies and process development of clinical-scale manufacturing of trem-cel. Preclinical data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function. At clinical scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function. Trem-cel pharmacology studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing. Toxicology assessment revealed no adverse findings, and no significant or reproducible off-target editing events. Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin in vitro and in vivo. These studies supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).

Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by abnormal clonal expansion and is the most common form of adult acute leukemia. Though hematopoietic stem cell transplant is the standard of care for high-risk AML patients, relapse post-transplantation occurs in 40% of these patients, highlighting the need for new therapeutic approaches such as immunotherapy. Flow cytometric profiling of 26 AML patient samples demonstrated that CD33 is expressed on >94% of blasts/LSCs (leukemic stem cells) while CLL-1 is expressed on >85% of blasts/LSCs at density levels targetable by immunotherapies (1283-2260 molecules per blast), suggesting that immuno-targeting both CD33 and CLL-1 can address AML heterogeneity and reduce chances of tumor resistance. Targeting these antigens, however, can lead to cytopenia due to shared expression on normal hematopoietic cells. Aims: We propose to delete both CD33 and CLL-1 from hematopoietic stem cell grafts, thereby restricting these antigens to AML cells only to enable subsequent immunotherapy without risk of on-target off-tumor toxicities. Methods: Mobilized peripheral blood CD34+ human hematopoietic stem and progenitor cells (hHSPCs) were gene edited using CRISPR/Cas9 with gRNAs against CD33 and CLL-1, resulting in biallelic deletion of both genes in >80% cells. Results: The viability of multiplex-edited hHSPCs was >90% with no impact on the distribution of hematopoietic stem cells, multi-potent progenitors, myeloid or lymphoid progenitors. The editing frequencies were maintained in each of these HSPC compartments. Loss of CD33 and CLL-1 proteins did not impact myeloid and erythroid potential or differentiation into granulocytic or monocytic lineages in vitro. Additionally, myeloid cells derived from multiplex-edited hHSPCs also retained their function, demonstrating similar phagocytotic capacity and cytokine secretion compared to unedited control cells. Multiplex-edited hHSPCs xenotransplanted into NOD-scid IL2Rgammanull (NSG) mice showed no defect in long-term engraftment. No impact was observed in lymphoid and myeloid lineage reconstitution (10 hematopoietic lineages analyzed) while CD33 and CLL-1 antigens were simultaneously absent from >95% of cells due to genome editing (CD33+/CLL-1+ monocytes: 0.9±0.4% for multiplex-edited and 71.5±1.4% unedited arms; CD33-/CLL-1- monocytes: 92.2±2.5% for multiplex edited and 1.1±0.3% unedited arms). This data highlights the long-term persistence of high levels of biallelic editing at both genes with no impact to myeloid differentiation in vivo. Quantification of on-target editing by NGS amplicon sequencing revealed no loss of total editing frequencies after engraftment and most importantly, no detectable translocations due to our temporally staggered editing strategies to reduce simultaneous double stranded breaks at both loci. Together, these pre-clinical data indicate that gene modifications in dual engineered cells can persist long-term post-engraftment and that there were no counterselection for these cells. Lastly, CD33 and CLL-1 dual edited cells also showed significant protection from CD33 and/or CLL-1 CAR-T cells. Summary/Conclusion: Here we show that CD33 and CLL-1 multiplex edited hHSPCs maintain robust hematopoiesis with high levels of editing at both loci. Pairing multiplex-edited hHSPCs with subsequent multi-specific immunotherapy can obviate concerns around tumor heterogeneity and escape mechanisms related to single antigen downregulation, transforming the current treatment approach for AML.

Acute myeloid leukemia (AML) is a clinically heterogeneous disease characterized by chromosomal abnormalities, gene mutations, and inter- and intra-patient genetic and phenotypic variability. Therapies specifically targeting cell surface antigens are promising treatments for AML, but surface antigen expression variability and potential antigen escape mechanisms may impact efficacy of single-targeting therapies. Delineating surface antigen expression uniformity and density between patients and during disease progression from diagnosis to relapse will enable rational prioritization of AML antigens for adaptive and combinatorial targeting. Here, we elucidate surface antigen expression patterns for candidate therapeutic targets by performing multimodal integrative analysis of AML surface proteins and transcriptomes of matched diagnosis and relapse clinically annotated samples. To identify potential therapeutic AML targets, we compiled a comprehensive list of protein surface targets reported in clinicaltrials.gov, literature, and large proteomics mass spectrometry datasets of AML patient bone marrow samples (deBoer et. al. Cancer Cell 2018; Jayavelu et al, Cancer Cell 2022). To focus on surface antigens and reduce the risk of targeting antigens that may have off-tumor solid tissue toxicity, we then excluded antigens that lack plasma cell membrane localization or are expressed on healthy non-hematologic tissues. We adapted Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) to generate longitudinal single-cell multimodal readouts of both the transcriptome and targeted surface proteins of matched diagnostic and relapsed samples from 42 adults with AML (Figure 1). Antibody derived tags (ADTs) against selected antigens were used at saturating conditions. Single cell libraries were prepared with 10X Genomics 5' Feature Barcoding Library Kit v2 and sequenced on Illumina NextSeq 2000. Cellranger v6.0.0 and Seurat v3 were used for mapping, read quantification, and analysis, respectively. We leveraged AML cell lines with known expression of benchmark antigens to assess the accuracy of the ADT readout by correlating it with antibody bound per cell (ABC) values derived from the traditional flow cytometry-based QuantiBRITE assay. Patient bone marrow data were projected onto low dimensional space using UMAP to visualize the population structure of the data. We observed separation of AML blasts from healthy cell lineages and delineation of blast gating based on CD45 expression. k-nearest neighbors clustering identified phenotypically diverse clusters within AML blasts within each patient sample, enabling quantification of specific antigen-positive clusters and antigen co-expression patterns. We found strong correlation between ADT readout and corresponding ABC values for cell surface protein expression on AML blasts of our benchmark antigens (CD33, CD123, CLL1, and ADGRE2). We then proceeded to systematically rank 87 AML surface antigens in blast populations based on ADT readout. Moreover, transcriptome profiling allowed investigation of antigens with limited antibody availabilities, exploration into novel cell states, and identification of signaling pathways that may associate with the presence of particular surface antigens. Identification and analysis of leukemic stem cells are ongoing. Healthy bone marrow and peripheral blood will be analyzed alongside AML samples to elucidate antigen target expression during normal hematopoiesis. In summary, our multimodal analysis of AML surface antigens informs cell surface clonal heterogeneity of patient AML and builds a reference atlas of expression patterns and intensities of potential surface AML antigen targets. In ongoing work, we are analyzing correlations between clonal surface antigen expression in longitudinal analysis of diagnosis and relapse samples as well as correlation with clinical parameters. This comprehensive surface antigen expression profiling will inform selection of therapeutically targetable and consistently expressed antigens to address AML heterogeneity in future immunotherapy targeting studies. Note: J.E. and M.U. share co-first authorship. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.

TET2 is among the most frequently mutated genes in hematopoietic malignancies. Inactivating mutations in TET2 are found in ∼30% of patients with myelodysplastic syndrome (MDS), ∼19% with de novo acute myeloid leukemia (AML), and ~15% with myeloproliferative neoplasm (MPN). TET2 mutations are also identified in a subset of individuals over 50 years of age with clonal hematopoiesis of indeterminate potential (CHIP), a condition that predisposes affected individuals to progression to myeloid malignancy and atherosclerotic heart disease with heart attack or stroke. TET2 mutations represent an early genetic lesion in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state of clonal dominance that predisposes to the acquisition of additional mutations. Thus, effective therapy for patients with TET2 mutations in HSPCs will require identifying drugs that are selectively lethal to TET2 mutant HSPCs but spare normal HSPCs, a property analogous to the widely studied genetic relationship called “synthetic lethality”. Using a tet2-mutant zebrafish model created in our laboratory, we screened for drugs from libraries of FDA-approved compounds and drugs in Phase I/II testing. We found that nuclear exporter XPO1 inhibitors, selinexor (KPT-330) and eltanexor (KPT-8602), were among the most promising, in that they are selectively lethal to HSPCs in tet2-mutant compared to wild-type fish. Moreover, we treated HSPCs of wild-type and Tet2-deficient mice with both selinexor and eltanexor in a methylcellulose colony formation assay. WT HSPCs lose their replating capacity at passage 3 (P3), while Tet2-mutant clones demonstrate aberrant sustained self-renewal capacity over multiple replatings. We found that both drugs selectively kill primary Tet2-mutant murine HSPCs and also block the aberrant self-renewal of these cells. By transplanting WT and Tet2-mutant CD45.2 donor cells into CD45.1 recipient mice, we monitored peripheral blood cells before and after treatment with selinexor or eltanexor to explore synthetic lethality of these drugs in vivo. Moreover, the human AML cell line K562 with TET2 homozygous mutations generated with CRISPR-Cas9, was more sensitive to selinexor and eltanexor than the parental cell line. To elucidate the mechanism behind the selective targeting of Tet2-mutant blood stem cells, we will use PRO-Seq and START-Seq methods to identify transcriptional elongation and initiation sites at single-nucleotide resolution. These preclinical studies must be done to show a therapeutic efficacy and mechanism of action of these new drugs before they can be translated to improve therapy of patients who have TET2 inactivating mutations. Citation Format: Nicole Prutsch, Chang-Bin Jing, Julia Etchin, Alla Berezovskaya, Hong Tiv, Michael J. Poitras, Prafulla Gokhale, Yosef Landesman, A. Thomas Look. Selective activity of XPO1 inhibitors in TET2-mutant myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2953.