Satoshi Kaito

Supplementary Figure 1. Activation status of anti-U5 snRNP200 CAR T cells co-cultured with AML cells in vitro. Supplementary Figure 2. Efficacy of IL-18 armored anti-U5 snRNP200 CAR T cells and their dependency on IFNg secretion. Supplementary Figure 3. Efficacy of IL-18 armored anti-U5 snRNP200 CAR T cells in AMLPDX models. Supplementary Figure 4. Evaluation of IL-18 armored U5 snRNP200 CAR T cells in pediatric AML PDXs. Supplementary Figure 5. Safety assessment of anti-U5 snRNP200 CAR T cells. Supplementary Figure 6. Efficacy of mouse IL-18 secreting U5 snRNP200 CAR T cells in a syngeneic mouse AML model. Supplementary Figure 7. UMAPs of bone marrow cells annotated by cell type and marker gene expression in cell type. Supplementary Figure 8. Clonal expansion of endogenous T cells in mice treated with IL18 secreting anti-U5 snRNP200 CAR T cells. Supplementary Figure 9. Evaluation of mechanism underlying AML re-challenge in immunocompetent mice following AML treatment with U5 snRNP200 CAR T cells. Supplementary Figure 10. Surface expression of U5 snRNP200 and efficacy of anti-U5 snRNP200 CAR T cells in B-ALL. Supplementary Figure 11. Anti-leukemic activity of U5 snRNP200 against B-ALL in vivo.

Supplementary Table S1. Clinical and genetic characteristics of AML and B-ALL patients for PDX models. Supplementary Table S2. TCR sequences of bone marrow T cells in syngeneic AML mouse model. Supplementary Table S3. Clinical and genetic characteristics of B-ALL patients for evaluation of surface U5 snRNP200 expression.

Developing chimeric antigen receptor (CAR) T cells for acute myeloid leukemia (AML) has been challenging due to a lack of known AML-associated antigens that spare normal hematopoietic precursor cells. Here we reasoned that donor autoantibodies from AML recipients cured following allogeneic transplant and responsible for graft-versus-leukemia effect could be engineered to create effective CAR-T cells. We generated CAR-T cells against one such antigen - U5 snRNP200, an RNA helicase localized to the AML cell surface and absent from normal hematopoietic precursors. Anti-U5 snRNP200 CAR-T cells were effective in human and syngeneic models of AML as well as B-cell acute lymphoblastic leukemia (B-ALL), a setting where surface U5 snRNP200 is also present. Armoring CAR-T cells with IL-18 led to antigen gain on AML, durable remission, and protection from AML rechallenge. These data thereby identify a CAR-T cell platform which addresses prior limitations in tumor-selectivity and safety for patients with acute leukemias.

The biological mechanisms that sustain the vast blood production required for healthy life remain incompletely understood. To search for cell intrinsic regulators of hematopoiesis, we perform a genome-wide in vivo hematopoietic stem and progenitor cell (HSPC)-based CRISPR knockout screen. We discover SAGA complex members, including Tada2b and Taf5l , as key regulators of hematopoiesis. Loss of Tada2b or Taf5l strongly inhibits hematopoiesis in vivo, causing a buildup of immature hematopoietic cells in the bone marrow. The SAGA complex deposits histone H3 lysine 9 acetylation (H3K9ac) and removes histone H2B ubiquitination (H2Bub). Loss of Tada2b leads to a reduction in H3K9ac levels and altered H2Bub enrichment in HSPCs, implicating disruption of SAGA complex activity. This is associated with upregulation of interferon pathway genes, reduced mitochondrial activity, and increased megakaryocyte progenitor cell commitment. Loss of these factors also enhances the cell outgrowth and the interferon pathway in an in vivo human myelodysplastic syndrome cell line model. In summary, this study identifies the SAGA complex as an important regulator of hematopoiesis.

Myelodysplastic syndrome (MDS) originates from hematopoietic stem cell (HSC) clones with acquired gene mutations. However, the molecular characteristics of MDS stem cells remain poorly understood. Here, we show that the chromatin accessibility profiles of MDS stem cells more accurately reflect disease status than those of progenitor cells and reveal the process of stem cell alterations during disease progression. Characterization of differentially accessible regions (DARs) shows that MDS stem cells acquire progenitor-like chromatin accessibility during disease progression, leading to disruption of the normal stem-progenitor hierarchy. Profiling of transcription factor-binding motifs at DARs further uncovers precocious activation of myeloid transcriptional networks in MDS stem cells, with a concurrent loss of HSC-associated regulatory programs. In particular, increased chromatin accessibility at CEBP target sites represents the myeloid reprogramming status of MDS stem cells. Newly developed “progenitor scores” based on chromatin accessibility stratify disease status and correlate well with prognosis. These findings indicate that chromatin landscapes of MDS stem cells define their cell-autonomous behavior and contribute to disease progression.

Supplementary Table S1: Patient information, Table S2: DEGs upregulated in CD138+ and CD138− MM cells detected in bulk RNA-seq, Table S3: DEGs in each cluster detected in scRNA-seq, Table S4: List of sgRNAs used in CRISPR/Cas9 screening, Table S5: List of candidate genes from CRISPR/Cas9 screening, Table S6: List of differential splicing events detected in bulk RNA-seq, Table S7: List of differential splicing events detected in scRNA-seq, Table S8: List of differential splicing events detected in RNA-seq of RBM39 KO and indisulam-treated AMO-1 cells, and Table S9: Primer sequences for amplification and sequencing of sgRNA regions, sgRNAs for CRISPR/Cas9 KO and RT-PCR primers to confirm splicing patterns.

Fig. S1: Sub-fractionation of MM cells by flow cytometry, Fig. S2: Detection of clonal repertoire in MM subfractions, Fig. S3: Heterogeneity of MM cells and cell surface marker gene expression, Fig. S4: DEGs in CD138+ and sCD138− MM cells, Fig. S5: Single-cell RNA-seq analysis of MM cells, Fig. S6: Single-cell RNA-seq and VDJ-seq analysis of MM cells, Fig. S7: Aberrant splicing in therapy-resistant sCD138− MM cells, Fig. S8: Re-analysis of published scRNA-seq data of BM MNCs of MM patients, Fig. S9: Aberrant splicing induced by RBM39 inhibition, and Fig. S10: RBM39 overexpression serves as a therapeutic target.

The molecular basis of therapy resistance in multiple myeloma remains poorly understood. In this study, we performed single-cell RNA sequencing coupled with VDJ-targeted sequencing of highly purified primary multiple myeloma cells from patient bone marrow. This approach uncovered cellular heterogeneity and phenotypic plasticity of multiple myeloma cells across a spectrum of CD138 expression, accompanied by drastic epigenetic alterations. Notably, therapy-resistant subpopulations were identified within a minor fraction of CD138– multiple myeloma cells, which were shown via CRISPR/Cas9 screening to be vulnerable to splicing pathway inhibition. Consistently, this fraction of CD138– multiple myeloma cells showed increased differential splicing associated with overexpression of SR protein family splicing factors. Among these splicing factors, RBM39 was overexpressed in therapy-resistant cells and involved in aberrant splicing. Both genetic and pharmacologic RBM39 inhibition exhibited a significant lethal effect on multiple myeloma cells. Collectively, our findings identify distinct therapy-resistant multiple myeloma subpopulations and highlight targeting the splicing pathway as a promising therapeutic strategy. Significance: Single-cell RNA sequencing coupled with VDJ-targeted profiling identified distinct therapy-resistant subpopulations within the minor CD138– fraction of multiple myeloma cells. These subpopulations were characterized by increased differential splicing events associated with overexpression of splicing factors from the SR protein family, with CD138− cells showing selective vulnerability to pharmacologic targeting of the splicing factor RBM39. See related commentary by Maron and Abdel-Wahab, p. 535

Post-translational modification (PTM) of histones dynamically regulates gene transcription and is closely related to cancer development. Altering the epigenome is thus a promising strategy for cancer chemotherapy. However, there have been no methods to artificially introduce physiologically relevant histone PTMs and their transcription profile without genetic engineering in living cells. Here we report a cell-permeable histone acetylation catalyst, BAHA-LANA-PEG-CPP44, enabling cancer epigenome manipulation. The catalyst selectively entered leukemia cells, bound to chromatin, and selectively acetylated H2BK120 of endogenous histones in a short reaction time. The catalytic histone acetylation attenuated chromatin binding of negative elongation factor E (NELFE), an RNA polymerase II regulatory factor, and changed the transcription profile in leukemia cells. The in-cell chemical catalysis slowed proliferation of leukemia cells and reduced their tumorigenic potential in mice. As this approach requires no genetic engineering and is orthogonal to canonical epigenetic drugs, it may represent a new modality of cancer chemotherapy.