Markus Muschen
University of California, San Francisco, Laboratory Medicine, Faculty Member
SUMMARY More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define... more
SUMMARY More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.
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Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role... more
Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role for PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small-molecule inhibition of PTEN in human pre-B ALL cells resulted in hyperactivation of AKT, activation of the p53 tumor suppressor cell cycle checkpoint and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre-B cell receptor signaling, which engaged a deletional checkpoint for the removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for the elimination of autoreactive B cells and represents a new strategy to overcome drug resistance in human ALL.
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Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate... more
Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic Casein Kinase II (CK2) and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes resulting in an anti-leukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia (B-ALL) xenografts as ...
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Recent studies have linked aberrant B-cell activation in the context of aberrant immune responses to infectious pathogens to malignant transformation and development of leukemia and lymphoma. A new study in this issue demonstrates that... more
Recent studies have linked aberrant B-cell activation in the context of aberrant immune responses to
infectious pathogens to malignant transformation and development of leukemia and lymphoma. A new study in
this issue demonstrates that common infections can be drivers of clonal evolution of premalignant B-cell precursors
toward childhood leukemia.
infectious pathogens to malignant transformation and development of leukemia and lymphoma. A new study in
this issue demonstrates that common infections can be drivers of clonal evolution of premalignant B-cell precursors
toward childhood leukemia.
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Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by... more
Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.
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Hodgkin and Reed Sternberg (H-RS) cells represent the malignant cells in classical Hodgkin's disease. Although derived from germinal center B cells, they do not express surface immunoglobulin. This has been explained by the presence... more
Hodgkin and Reed Sternberg (H-RS) cells represent the malignant cells in classical Hodgkin's disease. Although derived from germinal center B cells, they do not express surface immunoglobulin. This has been explained by the presence of crippling mutations within the immunoglobulin genes in numerous cases of Hodgkin's disease. As immunoglobulin gene expression in B cells requires an interaction between octamer sites and the transactivating factors Oct-2 and Bob-1, this study addresses the expression of the transcription factors Oct-2 and Bob-1 in H-RS cells. In Hodgkin's disease-derived cell lines, low levels of Oct-2 transcripts but no Oct-2 protein were detected. Transcripts of Bob-1, a B-cell-specific co-factor of Oct-2, could not be observed in these cell lines. Absence of Oct-2 and Bob-1 protein expression in primary H-RS cells was demonstrated by performing immunohistochemistry in 20 cases of classical Hodgkin's disease. H-RS cells stained negative for both prot...
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Expression of CD95 (Apo-1/Fas) ligand and its two receptor isoforms, in response to all-trans retinoic acid and interferon gamma (IFNgamma), was analyzed atthe mRNA and protein levels in human Tera-2 embryonal carcinoma cells. Exposure of... more
Expression of CD95 (Apo-1/Fas) ligand and its two receptor isoforms, in response to all-trans retinoic acid and interferon gamma (IFNgamma), was analyzed atthe mRNA and protein levels in human Tera-2 embryonal carcinoma cells. Exposure of Tera-2 cells to all-trans retinoic acid for up to 16 days led to a decrease of CD95 ligand expression when compared to the control conditions, whereas expression of both CD95 isoforms increased. These changes were functionally significant since Tera-2 cells treated with all-trans retinoic acid for six to 16 days were more susceptible to CD95-mediated apoptosis. On the other hand, Tera-2 cells lost their capacity to induce apoptosis in CD95 receptor bearing Jurkat T lymphocytes after six days of incubation with all-trans retinoic acid. When Tera-2 cells were treated with IFNgamma, expression of CD95 ligand and both CD95 receptor isoforms increased within 24 hours. Tera-2 cells were then more susceptible to CD95 mediated apoptosis but also killed mor...
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The outcome for pediatric ALL patients that relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale shRNA... more
The outcome for pediatric ALL patients that relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale shRNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of this data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the MAPK pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrated that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and p...
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In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B... more
In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34(+) HSCs and CD10(+) CD19(+) PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might...
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Despite recent advances in the cure rate of acute lymphoblastic leukaemia (ALL), the prognosis for patients with relapsed ALL remains poor. Here we identify FOXM1 as a candidate responsible for an aggressive clinical course. We show that... more
Despite recent advances in the cure rate of acute lymphoblastic leukaemia (ALL), the prognosis for patients with relapsed ALL remains poor. Here we identify FOXM1 as a candidate responsible for an aggressive clinical course. We show that FOXM1 levels peak at the pre-B-cell receptor checkpoint but are dispensable for normal B-cell development. Compared with normal B-cell populations, FOXM1 levels are 2- to 60-fold higher in ALL cells and are predictive of poor outcome in ALL patients. FOXM1 is negatively regulated by FOXO3A, supports cell survival, drug resistance, colony formation and proliferation in vitro, and promotes leukemogenesis in vivo. Two complementary approaches of pharmacological FOXM1 inhibition-(i) FOXM1 transcriptional inactivation using the thiazole antibiotic thiostrepton and (ii) an FOXM1 inhibiting ARF-derived peptide-recapitulate the findings of genetic FOXM1 deletion. Taken together, our data identify FOXM1 as a novel therapeutic target, and demonstrate feasibil...
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Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with... more
Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with the putative long non-coding RNA gene STL. Linking STL properties to leukemia has so far been difficult. Here, we describe a novel gene, OSTL (annotated as RNF217 in Genbank), which shares the first exon and a CpG island with STL but is transcribed in the opposite direction. Human RNF217 codes for a highly conserved RING finger protein and is mainly expressed in testis and skeletal muscle with different splice variants. RNF217 shows regulated splicing in B cell development, and is expressed in a number of human B cell leukemia cell lines, primary human chronic myeloid leukemia, acute myeloid leukemia with normal karyotype and acute T-ALL samples. Using a yeast two-hybrid screen, we identified the anti-apoptotic protein HAX1 to interact with RNF217....
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Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the... more
Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the H/RS cells from apoptosis. Given the importance of CD95 (Apo-1/Fas)-mediated apoptosis for negative selection within the germinal center, single micromanipulated H/RS cells from 10 cases of cHD were analyzed for somatic mutations within the CD95 gene. Three clonal mutations within the 5' regions were amplified from single H/RS cells in one case. From H/RS cells of another case, two mutations within the last exon coding for the death domain were detected. About half of these H/RS cells carried a monoallelic stop-codon; the remaining tumor cells harbored a monoallelic replacement mutation. Both mutations likely impair CD95 function. Because all these H/RS cells also bear clonal mutations inactivating the IkappaB alpha gene, the IkappaB alpha mutation...
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Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG... more
Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones are accessible. We also examine in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones are found to be more accessible relative to the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more refined models for why these zones are 100 to 1000-fold more prone to undergo chromosomal translocation than the adjacent regions.
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... Abrahamsson AE, Geron I, Gotlib J, Dao KH, Barroga CF, Newton IG, Giles FJ, Durocher J, Creusot RS, Karimi M, Jones C, Zehnder JL ... Genes Dev 15:1688–1705 Mikesch JH, Steffen B, Berdel WE, Serve H, Müller-Tidow C (2007) The emerging... more
... Abrahamsson AE, Geron I, Gotlib J, Dao KH, Barroga CF, Newton IG, Giles FJ, Durocher J, Creusot RS, Karimi M, Jones C, Zehnder JL ... Genes Dev 15:1688–1705 Mikesch JH, Steffen B, Berdel WE, Serve H, Müller-Tidow C (2007) The emerging role of Wnt signaling in the ...
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ABSTRACT
Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's dis- ease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the... more
Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's dis- ease (cHD) are thought to be derived from preapoptotic germinal center B cells. However, little is known about the transforming events rescuing the precursor of the H/RS cells from apoptosis. Given the importance of CD95 (Apo-1/Fas)-mediated apoptosis for negative selection within the germinal center, single micromanipulated H/RS cells from 10 cases
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At the pre-B cell receptor (BCR) checkpoint, developing pre-B cells are selected for successful rearrangement of V(H)-DJ(H) gene segments and expression of a pre-BCR. Reduced stringency at this checkpoint may obstruct the B cell... more
At the pre-B cell receptor (BCR) checkpoint, developing pre-B cells are selected for successful rearrangement of V(H)-DJ(H) gene segments and expression of a pre-BCR. Reduced stringency at this checkpoint may obstruct the B cell repertoire with nonfunctional B cell clones. Earlier studies have described that activation of B cell lymphoma/leukemia (BCL)6 by a functional pre-BCR mediates positive selection of pre-B cells that have passed the checkpoint. This concept is now further elaborated by the recent finding that the BTB and CNC homology 1 basic leucine zipper transcription factor 2 (BACH2) induces negative selection and opposes BCL6 function prior to the pre-BCR checkpoint. Here, we discuss the antagonism between BCL6 and BACH2 during early B cell development, as well as its implications in both repertoire selection and counter-selection of premalignant clones for leukemia suppression.
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ABSTRACT Background. Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1)... more
ABSTRACT Background. Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. Methods. In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). Results. in the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, how ever, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. Conclusion. This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.
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Research Interests: Design Research, Gamma Rays, Apoptosis, Multidisciplinary, Cell Differentiation, and 12 moreHumans, Mutation, Alternative splicing, Alternative Splicing, Base Sequence, B Cell Development, Cell Survival, Full Length Movies, Internal ribosome entry site, Acute Lymphoblastic Leukemia, Tyrosine Kinase, and Gamma radiation
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B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre-B cell receptor-dependent stages. The Philadelphia chromosome-positive (Ph(+)) subtype of ALL accounts for... more
B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre-B cell receptor-dependent stages. The Philadelphia chromosome-positive (Ph(+)) subtype of ALL accounts for 25-30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre-B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph(+) ALL cells. Pre-B cell receptor-mediated cell cycle arrest in Ph(+) ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre-B cell receptor signaling pathway, even if expression of the pre-B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre-B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph(+) ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre-B cell receptor-mediated tumor suppression.
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Research Interests: Flow Cytometry, Apoptosis, Sequence Analysis, Signal Transduction, Humans, and 12 moreMutagenesis, Polymerase Chain Reaction, Molecular cloning, Tumor Suppressor Gene, Experimental Medicine, Negative Selection Algorithm, B Lymphocytes, Side Effect, Immunoglobulin, Germinal Center, Death Domain, and Somatic Hypermutation
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Research Interests: Flow Cytometry, Western blotting, RNA interference, Humans, Sequence alignment, and 12 moreMutation, The, Tumor Suppressor Gene, Experimental Medicine, B Lymphocytes, Base Sequence, DNA binding proteins, Philadelphia Chromosome, Acute Lymphoblastic Leukemia, Oligonucleotides, Germinal Center, and Somatic Hypermutation
Research Interests: Stem Cells, Flow Cytometry, Stem Cell, Western blotting, Cell Division, and 30 moreHematopoietic Stem Cells, Hippocampus, Cell line, Cell Differentiation, Humans, Cell fusion, Placenta, Sheep, Femur, Cord Blood, Animals, Polymerase Chain Reaction, Astrocyte, Animal Model, The, Rat Brain, Cell Transplantation, Phenotype, Calcium Phosphate, Rats, Myocardium, Sodium channel, Time Factors, Adipocytes, Experimental Medicine, Wistar Rats, Immunophenotyping, Osteoblasts, Bone and Bones, and Nude mice
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B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR)1 or hyperactivation above maximum (for example, self-reactive BCR)2,3... more
B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR)1 or hyperactivation above maximum (for example, self-reactive BCR)2,3 thresholds of signalling strength causes negative selection. In 25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), whichmimics constitutively active pre-BCR signalling4,5. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival6. We tested the hypothesis that targeted hyperactivation—above a maximum threshold—will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR–ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patientderived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6
(ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYKand engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
(ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYKand engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.