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 three 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 a reconsideration of the role of HIF-1α and, as a consequence, question its generic therapeutic usefulness in AML

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.publishedVersionPeer reviewe

Molecular Interrogation and Functional Studies of Acute Leukemia

Hematological malignancies are defined by their underlying genetic alterations, many of which are used to diagnose patients to classify them to different risk groups that dictate the therapy given. Recent advances in high-throughput sequencing have highlighted the presence of co-occurring genetic lesions and that they may form distinct genetic clones that evolve throughout disease progression. Acute leukemia is a group of diseases affecting either the lymphoid or myeloid lineage in hematopoiesis, resulting in acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). Certain genetic alterations are closely tied to specific leukemia types, while others are more promiscuous. In this thesis, we have used high-resolution genome-wide methods and murine models to study leukemia as a way to increase our knowledge how leukemia arises and best can be treated.In the first study (Article I) we characterized the genetic alterations in a case presenting with a rare myelodysplatic/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U) that later progressed to AML. Through comprehensive analyses of the MDS/MPN-U and AML samples, we observed that all genetic lesions detected at AML diagnosis were present already at the MDS/MPN-U stage, likely in a similar clonal composition. Further, targeted drug analysis of the AML sample suggested clinically approved drugs from which the patient could benefit at a potential relapse.Genetic rearrangements of the epigenetic regulator KMT2A (KMT2A-R) often co-occur with activating mutations in genes involved in intracellular signaling. In the second study (Article II) we show that mutations in FLT3 and NRAS significantly accelerate KMT2A-R driven AML onset, even when present in a subclone as exemplified by the FLT3N676K mutation. The presence of an activating mutation affected the leukemias transcriptional profiles by further enhancing transcriptional programs previously associated with KMT2A-Rs. Genomic characterization of mouse leukemias unveiled de novo signaling mutations in several mice harboring only a KMT2A-R, emphasizing the importance of such mutations in KMT2A-R leukemogenesis. KMT2A-Rs occur in both ALL and AML but the molecular and/or biological mechanisms determining the lineage affiliation remain largely elusive for this disease. In the third study (Article III) we demonstrated the that FLT3N676K promote myeloid expansion of KMT2A-R leukemia in primary human cells. We further showed that established KMT2A-R ALL and AML cells displayed expression profiles closely linked to their respective lineage but that these cells still display a certain immunophenotypic plasticity.Previously, a large portion of pediatric B-cell precursor ALL (BCP-ALL) patients could not be classified to any of the established molecular subtypes. Chromosomal alterations are a hallmark of BCP-ALL and in the last study (Article IV) we employed high-throughput sequencing to define the fusion gene landscape of 195 pediatric BCP-ALL. Besides identifying several novel in-frame fusion genes, we also described two new oncogenic leukemia subtypes. These two subtypes were associated with distinct genetic lesions, including genetic rearrangements of the DUX4 gene and genetic alterations of ETV6 and IKZF1. Taken together, the work included in this thesis highlights the major impact that specific genetic alterations have on leukemogenesis, and how their autonomous and non-autonomous cooperation influence clonal evolution, disease phenotype, and molecular profiles of the leukemia

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 N79QN116Q 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

Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells

[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 we call SyNthetic Intramembrane Proteolysis Receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the potential transformative utility 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.]https://www.biorxiv.org/content/10.1101/2021.05.21.445218v1Published versio

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

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