Bingyi Chen

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.

Leukemia is a malignant tumor with a high recurrence rate and poor prognosis for patients. Thus, there is an urgent need to explore new therapeutic targets that play critical roles in leukemogenesis but have little effect on normal hematopoietic cells. Here, we show that RNA binding protein with multiple splicing (RBPMS), which is highly expressed in acute myeloid leukemia (AML) and associated with poor prognosis of AML, plays critical roles in leukemogenesis. Our study shows that inhibition of RBPMS inhibits self-renewal of leukemia-initiating cells (LICs) and leukemia development but has little effect on normal hematopoiesis. Mechanistically, RBPMS recruits the N ⁶ -methyladenosine (m ⁶ A) reader insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which promotes the stability of the forkhead box O1 ( FOXO1 ) mRNA in an m ⁶ A-dependent manner. Moreover, RBPMS contributes to the progression of leukemia by directly binding to FOXO1 and promoting FOXO1-regulated glycolysis. Overexpression of FOXO1 has been shown to reverse RBPMS inhibition–induced phenotypes in both leukemic cells and mouse models. We also designed a specific inhibitor of RBPMS that has therapeutic effects in AML patient-derived xenograft (PDX) models. We therefore highlight RBPMS as a promising drug target for leukemia therapy.

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.

Patients with myelodysplastic syndrome (MDS) harboring SRSF2 (serine and arginine rich splicing factor 2) mutations exhibit poor prognosis and aberrant inflammatory activation, underscoring an urgent need for therapies. Here, we reveal that low messenger RNA expression of SETD2 ( SET domain containing 2 ) in hematopoietic stem and progenitor cells (HSPCs) from patients with MDS carrying SRSF2P95 mutations (SRSF2 ^P95-Mut MDS) correlates with adverse outcomes and increased inflammation. Multivariate analysis confirmed the correlation between low SETD2 expression and poor prognosis in patients with SRSF2 ^P95-Mut MDS. Furthermore, Setd2 loss in the Srsf2 ^P95H/+ mouse model resulted in lethal MDS with hyperinflammation and expansion of myeloid-derived suppressor cells (MDSCs). Mechanistically, SETD2 methylates SRSF2 ^P95H at lysine-17 and lysine-65 to inhibit aberrant splicing of CEACAM1-4 (isoforms of carcinoembryonic antigen cell adhesion molecule ), which enhances interleukin-1β (IL-1β) signaling through Slc7a11 (solute carrier family 7 member 11)–mediated cystine uptake, thereby promoting HSPC differentiation into MDSCs, establishing an IL-1β–driven immunosuppressive microenvironment. These findings identify the SRSF2 ^P95H K17 ^me1 K65 ^me2 –CEACAM1-4 signaling axis as a promising therapeutic target in SRSF2 ^P95-Mut MDS.

Supplemental Figure 1. Systematic domain-focused CRISPR screens of GAPs and GEFs and transcriptomic profiling identify ARHGAP45 as a hematologic malignancy-biased dependency linked to distinct genetic and risk subgroups. Supplemental Figure 2. ARHGAP45 is a selective dependency in hematopoietic malignancies, but dispensable for normal CD34+ hematopoietic stem/progenitor cells (HSPCs) in vivo. Supplemental Figure 3. ARHGAP45 regulates cell cycle and constrains AML differentiation. Supplemental Figure 4. ARHGAP45 suppresses RhoA activity, and hyperactivation of RhoA suppresses AML growth. Supplemental Figure 5. Loss of ARHGAP45 sensitizes AML to CDC42 inhibition. Supplemental Figure 6. Activity of CAR-like TCRs against ARHGAP45 derived HA-1H antigen in myeloid malignancies. Supplemental Figure 7. In vivo efficacy of CAR-like TCRs against AML cell lines or patient-derived xenograft (PDX) expressing HA-1H.

GTPase-activating proteins (GAP) and guanine nucleotide exchange factors (GEF) play key roles in cancer development, but their large number and potential redundancy have limited systematic evaluation. In this study, we perform unbiased genetic screens to identify GAPs and GEFs with cancer- and lineage-specific requirements, as well as dual perturbation screens to dissect functionally relevant interactors of GAPs and GEFs. Application to primary specimens from patients with acute myeloid leukemia uncovers the GAP ARHGAP45 as a targetable dependency shared across cancers of hematopoietic origin while being dispensable in normal hematopoiesis. We demonstrate that targeting ARHGAP45-expressing cells can be achieved through T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-encoded minor histocompatibility antigen and that pharmacologic targeting of CDC42 required upon ARHGAP45 depletion augments ARHGAP45-directed cell therapies. These studies provide a resource for probing oncogenic and druggable regulators of GTPases and strategies to target a GAP that represents a shared dependency across blood cancers.Significance:In this study, we systematically interrogated GAPs and GEFs in cancer and identified the GAP ARHGAP45 as a dependency shared across blood cancers while dispensable in normal hematopoiesis. Targeting ARHGAP45-expressing cells is achievable via T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-derived antigen and small-molecule inhibition of GTPases required upon ARHGAP45 loss.

Supplementary Table S1. Clinical sample information. Supplementary Table S2. Domain-focused sgRNA libraries used in this study and MAGeCK analysis of CRISPR screening results. Supplementary Table S3. sgRNA sequences. Supplementary Table S4. Gene sets used in this study. SupplementaryTable S5. TMT-based total proteomics and phospho-proteomics.

GTPase-activating proteins (GAP) and guanine nucleotide exchange factors (GEF) play key roles in cancer development, but their large number and potential redundancy have limited systematic evaluation. In this study, we perform unbiased genetic screens to identify GAPs and GEFs with cancer- and lineage-specific requirements, as well as dual perturbation screens to dissect functionally relevant interactors of GAPs and GEFs. Application to primary specimens from patients with acute myeloid leukemia uncovers the GAP ARHGAP45 as a targetable dependency shared across cancers of hematopoietic origin while being dispensable in normal hematopoiesis. We demonstrate that targeting ARHGAP45-expressing cells can be achieved through T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-encoded minor histocompatibility antigen and that pharmacologic targeting of CDC42 required upon ARHGAP45 depletion augments ARHGAP45-directed cell therapies. These studies provide a resource for probing oncogenic and druggable regulators of GTPases and strategies to target a GAP that represents a shared dependency across blood cancers. Significance: In this study, we systematically interrogated GAPs and GEFs in cancer and identified the GAP ARHGAP45 as a dependency shared across blood cancers while dispensable in normal hematopoiesis. Targeting ARHGAP45-expressing cells is achievable via T-cell receptor chimeric antigen receptor T cells directed at an ARHGAP45-derived antigen and small-molecule inhibition of GTPases required upon ARHGAP45 loss.