Allosteric SHP2 inhibition increases apoptotic dependency on BCL2 and synergizes with venetoclax in FLT3- and KIT-mutant AML.

Mutations in the receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although selective FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report the pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppresses proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that pharmacologic SHP2 inhibition unveils an Achilles' heel of RTK-driven AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in AML cell lines and in clinically relevant xenograft models. Our results provide mechanistic rationale and pre-clinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.

CD38 as a pan-hematologic target for chimeric antigen receptor T cells.

Many hematologic malignancies are not curable with chemotherapy and require novel therapeutic approaches. Chimeric antigen receptor (CAR) T-cell therapy is 1 such approach that involves the transfer of T cells engineered to express CARs for a specific cell-surface antigen. CD38 is a validated tumor antigen in multiple myeloma (MM) and T-cell acute lymphoblastic leukemia (T-ALL) and is also overexpressed in acute myeloid leukemia (AML). Here, we developed human CD38-redirected T cells (CART-38) as a unified approach to treat 3 different hematologic malignancies that occur across the pediatric-to-adult age spectrum. Importantly, CD38 expression on activated T cells did not impair CART-38 cells expansion or in vitro function. In xenografted mice, CART-38 mediated the rejection of AML, T-ALL, and MM cell lines and primary samples and prolonged survival. In a xenograft model of normal human hematopoiesis, CART-38 resulted in the expected reduction of hematopoietic progenitors, which warrants caution and careful monitoring of this potential toxicity when translating this new immunotherapy into the clinic. Deploying CART-38 against multiple CD38-expressing malignancies is significant because it expands the potential for this novel therapy to affect diverse patient populations.

Potent preclinical activity of FLT3-directed chimeric antigen receptor T-cell immunotherapy against FLT3- mutant acute myeloid leukemia and KMT2A-rearranged acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 or CD22 induce remissions in the majority of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), although relapse due to target antigen loss or downregulation has emerged as a major clinical dilemma. Accordingly, great interest exists in developing CAR T cells directed against alternative leukemia cell surface antigens that may help to overcome immunotherapeutic resistance. The fms-like tyrosine kinase 3 receptor (FLT3) is constitutively activated via FLT3 mutation in acute myeloid leukemia (AML) or wild-type FLT3 overexpression in KMT2A (lysine-specific methyltransferase 2A)-rearranged ALL, which are associated with poor clinical outcomes in children and adults. We developed monovalent FLT3-targeted CAR T cells (FLT3CART) and bispecific CD19xFLT3CART and assessed their anti-leukemia activity in preclinical models of FLT3-mutant AML and KMT2A-rearranged infant ALL. We report robust in vitro FLT3CART-induced cytokine production and cytotoxicity against AML and ALL cell lines with minimal cross-reactivity against normal hematopoietic and non-hematopoietic tissues. We also observed potent in vivo inhibition of leukemia proliferation in xenograft models of both FLT3-mutant AML and KMT2A-rearranged ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model pairing. We further demonstrate significant in vitro and in vivo activity of bispecific CD19xFLT3CART against KMT2Arearranged ALL and posit that this additional approach might also diminish potential antigen escape in these high-risk leukemias. Our preclinical data credential FLT3CART as a highly effective immunotherapeutic strategy for both FLT3- mutant AML and KMT2A-rearranged ALL which is poised for further investigation and clinical translation.

Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL.

Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.

Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies.

Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. SIGNIFICANCE: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.

Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design.

BACKGROUND: Successful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown. METHODS: In this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML. RESULTS: We observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A 'best-in-class' lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation. CONCLUSIONS: CD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML. TRIAL REGISTRATION NUMBER: clinicaltrials.gov; NCT03971799.