CD4+ CAR-T cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR-T cell therapy.

Multiple myeloma is characterized by frequent clinical relapses following conventional therapy. Recently, chimeric antigen receptor T (CAR-T) cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, while >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune intrinsic mechanisms may contribute to this resistance. While there were no pre-existing T cell features associated with clinical outcomes, we found that patients with a durable response to CAR-T cell treatment had greater persistence of their CAR-T cells compared to patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR-T cells. These cells expand in vivo early after infusion but express exhaustion markers (HAVCR2 and TIGIT) and remain polyclonal. Finally, we demonstrate that non-classical monocytes are enriched in the myeloma niche and may induce CAR-T cell dysfunction through mechanisms that include TGFß. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR-T cell therapy.

Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia.

Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3 D835H or NRAS G13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.

Modular design of synthetic receptors for programmed gene regulation in cell therapies.

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models.

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening "on-target/off-tumor" toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.

Paving New Roads for CARs.

Chimeric antigen receptor (CAR)-T cell therapy has had unprecedented impact in the treatment of hematological malignancies with few therapeutic options. However, it is clear that new strategies to enhance CAR-T cell function in solid tumors are needed to make these living drugs widely applicable. The roadblock in solid tumors has led to a surge in the development of strategies to enhance CAR-T cells through advanced receptor design, new tumor sensing mechanisms, coexpression of genes that improve T cell function or stimulate tumor immunity, and precise genome editing. Here we provide an overview of the current state of the art in CAR-T cell engineering and a framework for the development of next-generation immune cell therapies with synthetic biology.

De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia.

Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3 ITD , FLT3 N676K , and NRAS G12D accelerate KMT2A-MLLT3 leukemia onset. Further, also subclonal FLT3 N676K mutations accelerate disease, possibly by providing stimulatory factors. Herein, we show that one such factor, MIF, promotes survival of mouse KMT2A-MLLT3 leukemia initiating cells. We identify acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 in KMT2A-MLLT3 leukemia cells that favored clonal expansion. During clonal evolution, we observe serial genetic changes at the Kras G12D locus, consistent with a strong selective advantage of additional Kras G12D . KMT2A-MLLT3 leukemias with signaling mutations enforce Myc and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlight the importance of activated signaling as a contributing driver.

Identification of ETV6-RUNX1-like and DUX4-rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia.

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

Genomic profiling and directed ex vivo drug analysis of an unclassifiable myelodysplastic/myeloproliferative neoplasm progressing into acute myeloid leukemia.

Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs. © 2016 Wiley Periodicals, Inc.

HIF-1α can act as a tumor suppressor gene in murine acute myeloid leukemia.

Self-renewal of hematopoietic stem cells (HSCs) and leukemia-initiating cells (LICs) has been proposed to be influenced by low oxygen tension (hypoxia). This signaling, related to the cellular localization inside the bone marrow niche and/or influenced by extrinsic factors, promotes the stabilization of hypoxia-inducible factors (HIFs). Whether HIF-1α can be used as a therapeutic target in the treatment of myeloid malignancies remains unknown. We have used 3 different murine models to investigate the role of HIF-1α in acute myeloid leukemia (AML) initiation/progression and self-renewal of LICs. Unexpectedly, we failed to observe a delay or prevention of disease development from hematopoietic cells lacking Hif-1α. In contrast, deletion of Hif-1α resulted in faster development of the disease and an enhanced leukemia phenotype in some of the investigated models. Our results therefore warrant reconsideration of the role of HIF-1α and, as a consequence, question its generic therapeutic usefulness in AML.

The structural role of N-linked glycans on human glypican-1.

Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was reduced and that the protein did not accumulate inside the cells. Heparan sulfate substitution of N-glycosylation mutant N116Q was similar to wild-type glypican-1 while the N79Q mutant and also the double mutant N79Q,N116Q were mostly secreted as high-molecular-weight heparan sulfate proteoglycan. N-Glycosylation mutants and N-deglycosylated glypican-1 had far-UV circular dichroism and fluorescence emission spectra that were highly similar to those of N-glycosylated glypican-1. A single unfolding transition at high concentrations of urea was found for both N-deglycosylated glypican-1 and glypican-1 in which the N-glycosylation sites had been removed by mutagenesis when chemical denaturation was monitored by circular dichroism and fluorescence emission spectroscopy. In summary, we have found that the potential N-glycosylation sites in glypican-1 are invariably occupied and that the N-linked glycans on glypican-1 affect protein expression and heparan sulfate substitution but that correct folding can be obtained in the absence of N-linked glycans.