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Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell–mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4 , one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4 -null phenotype in vivo. We demonstrated that the Ypel4 -null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4 -null cells was diminished. Furthermore, Ypel4 -null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4 -null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4 -null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph⁺) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34⁺ cells and also in cord blood CD34⁺ cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph⁻) and leukemic (Ph⁺) cells within the CML CD34⁺CD38⁻ cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34⁺CD38⁻IL1RAP⁺ cells were Ph⁺, whereas CML CD34⁺CD38⁻IL1RAP⁻ cells were almost exclusively Ph⁻. By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph⁺ and Ph⁻ candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34⁺CD38⁻ cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph⁺ from Ph⁻ candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML.

Natural killer (NK) cells are large granular lymphocytes involved in our defense against certain virus-infected and malignant cells. In contrast to T cells, NK cells elicit rapid anti-tumor responses based on signals from activating and inhibitory cell surface receptors. They also lyse target cells via antibody-dependent cellular cytotoxicity, a critical mode of action of several therapeutic antibodies used to treat cancer. A body of evidence shows that NK cells can exhibit potent anti-tumor activity against chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS). However, disease-associated mechanisms often restrain the proper functions of endogenous NK cells, leading to inadequate tumor control and risk for disease progression. Although allogeneic NK cells can prevent leukemia relapse in certain settings of stem cell transplantation, not all patients are eligible for this type of therapy. Moreover, remissions induced by adoptively infused NK cells are only transient and require subsequent therapy to maintain durable responses. Hence, new strategies are needed to trigger full and durable anti-leukemia responses by NK cells in patients with myeloid malignancies. To achieve this, we need to better understand the interplay between the malignant cells, their microenvironment, and the NK cells. This review focuses on mechanisms that are involved in suppressing NK cells in patients with myeloid leukemia and MDS, and means to restore their full anti-tumor potential. It also discusses novel molecular targets and approaches, such as bi- and tri-specific antibodies and immune checkpoint inhibitors, to redirect and/or unleash the NK cells against the leukemic cells.

BackgroundPancreatic ductal adenocarcinoma (PDAC) represents a major clinical challenge due to its tumor microenvironment, which exhibits immune-suppressive properties that facilitate cancer progression, metastasis, and therapy resistance. Interleukin 1 (IL-1) signaling has been implicated as a driver in this process. Mechanistically, both IL-1α and IL-1β bind to the IL-1 receptor type 1, forming a complex with IL-1-receptor accessory protein (IL1RAP), which triggers downstream signaling pathways. The IL1RAP blocking antibody nadunolimab is currently in clinical development, but the precise consequences of inhibiting IL-1 signaling in PDAC remains elusive.MethodsTo evaluate the biological relevance of blocking IL1RAP using nadunolimab in a PDAC animal model, human PDAC cells and cancer-associated fibroblasts (CAFs) were co-transplanted into mice. To study the underlying mechanisms of IL1RAP blockade ex vivo, co-cultured PDAC cells and CAFs were treated with nadunolimab prior to RNA sequencing. Migration assays were performed to assess how nadunolimab affects interactions between CAFs and myeloid immune cells. Finally, to establish a clinical correlation between IL1RAP expression and nadunolimab treatment effects, we analyzed tumor biopsies from a clinical phase I/II study in which nadunolimab was administered to patients.ResultsIn the xenograft mouse model, nadunolimab exhibited antitumor effects only when human CAFs were co-transplanted with PDAC cells. IL-1 stimulation induced CAFs to secrete chemokines that recruited neutrophils and monocytes. The secretion of this chemokine and the migration of myeloid cells were inhibited by nadunolimab. Media conditioned by IL-1-stimulated CAFs sustained a neutrophil population with a tissue invasion phenotype, an effect that was reversed by nadunolimab. In a cohort of metastatic late-stage PDAC patients receiving nadunolimab as monotherapy, high IL1RAP expression in tumors was associated with extended progression-free survival.ConclusionsOur study demonstrates that targeting IL1RAP on CAFs inhibits IL-1-induced chemokine secretion and recruitment of neutrophils and monocytes, thereby counteracting the immunosuppressive microenvironment in PDAC. These findings highlight the therapeutic potential of targeting IL1RAP in PDAC.

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4 CRBN ), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4 CRBN . These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases. Lenalidomide, a derivative of thalidomide, is an effective drug for myelodysplastic syndrome; lenalidomide binds the CRL4 CRBN E3 ubiquitin ligase and promotes degradation of casein kinase 1a, on which the malignant cells rely for survival. Mechanism of action of lenalidomide Thalidomide was taken off the market when it was found to cause malformation in children whose mothers had taken it as a treatment for morning sickness in the late 1950s and early 1960s. Later it emerged that thalidomide and derivatives could be successfully used to treat certain haematopoietic disorders, and the thalidomide derivative lenalidomide has proved an effective therapy for myelodysplastic syndrome (MDS). Ben Ebert and colleagues now show why lenalidomide is particularly efficient in so-called del(5q) MDS — a frequent form of MDS carrying deletions in one copy of the chromosome 5q arm. They find that lenalidomide binds the CRL4 CRBN E3 ubiquitin ligase and promotes degradation of casein kinase 1α, which the malignant cells rely on for survival. In addition, a new analogue of thalidomide, CC-122, is shown to have greater potency than lenalidomide in inducing degradation of other CRBN substrates that are important in certain B cell malignancies.

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. In acute leukemia with KMT2A rearrangements ( KMT2A -R), activating signaling mutations are common. Here, the authors use a retroviral acute myeloid mouse leukemia model to show that subclonal de novo activating mutations drive clonal evolution in acute leukemia with KMT2A -R and enhance clonal fitness.

The secreted cysteine protease inhibitors, cystatin C and cystatin D, were added externally to leukemic U937, Jurkat, and HL‐60 cell cultures. The cystatins were internalized into endo‐lysosomal vesicles resulting in augmented hydrogen peroxide‐induced apoptosis, as well as decreased proliferation, both in apoptotic and nonapoptotic cells. Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL‐60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas‐induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase‐3‐like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V‐positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide‐induced apoptotic U937 cells with either cystatin C or D resulted in a dose‐dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1–9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL‐60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.

Michael Kharas and colleagues characterize the MSI2 protein interactome in leukemia cells and subsequently perform a functional screen identifying 24 genes required for leukemia in vivo . They focus on the RNA-binding protein SYNCRIP, showing that it regulates Hoxa9 and other transcripts involved in a myeloid leukemia stem cell program. The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.