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Extracellular matrix protein Matn4 regulates HSC stress response
Short Talk Presentations/Experimental Hematology 43 (2015) S45–S49
S45
Short Talk Presentations 2003 - RAPID FORMATION OF B CELL LEUKEMIA IN MICE EXPRESSING THE PAX5-JAK2 FUSION PROTEIN Sabine Jurado, Daniela Kostanova, and Meinrad Busslinger Research Institute of Molecular Pathology, Vienna, Austria
2005 - EXTRACELLULAR MATRIX PROTEIN MATN4 REGULATES HSC STRESS RESPONSE Hannah Uckelmann, Sandra Blaszkiewicz, and Marieke Essers DKFZ/HI-STEM, Heidelberg, Germany
Abnormalities in B cell development, regulation and survival can lead to immunodeficiency, autoimmunity and leukemia or lymphoma formation. Pax5 is considered to be the guardian of B cell identity; it is therefore not surprising that about a third of all B-cell precursor acute lymphoplastic leukemias (BCP-ALL) have heterozygous PAX5 alterations, including gene rearrangements. Among them are translocations that lead to the expression of fusion proteins, which are thought to interfere with normal Pax5 function. However, heterozygous Pax5 mutations are not sufficient for tumor formation in mice. Hence, a second oncogenic hit is required, such as the constitutive activation of STAT5 or deletion of CDKN2a/b. One recently described in-frame fusion protein, PAX5-JAK2, results in the loss of the auto-inhibitory pseudo-kinase domain of JAK2, potentially leading to a constitutively active JAK2 kinase (caJAK2). caJAK2 could in turn promote constitutive activation of STAT5 and therefore provide the second oncogenic hit necessary for leukemia formation. In order to determine the oncogenic potential of PAX5-JAK2, we have generated a mouse expressing this fusion protein from the Pax5 locus. Here we show that heterozygous expression of the Pax5-JAK2 allele in these mice, mimicking the clinical situation, is sufficient to drive leukemia formation within 12 weeks. Flow cytometric analyses of leukemic cells reveal the presence of surface markers corresponding to early stages of B-cell development, similar to the BCP-ALL diagnosis of human patients. Abnormal B cells can already be detected accumulating in the bone marrow of 4-5 week-old mice, and are later found in secondary lymphoid organs. Interestingly, these tumor cells have higher phosphoSTAT5 levels than normal B cells and can also undergo loss of expression of the wild-type Pax5 allele. In conclusion, the fast and reproducible leukemia formation observed in these mice makes it a faithful model to study the initiation and maintenance of leukemia of Pax5-JAK2 positive BCP-ALL, and current work is aimed at unraveling the mechanism of action of Pax5-JAK2.
Multipotent hematopoietic stem cells (HSCs) are found in a dormant state for most of their lifetime, which they exit only rarely to self-renew or differentiate to sustain blood supply. In acute stress situations such as infection, HSCs are activated to reconstitute compromised blood cells. The molecular and cellular mechanisms controlling stress-induced activation of HSCs remain largely unknown. The extracellular matrix adaptor protein Matrilin-4 (Matn4) is highly expressed in long-term HSCs (LT-HSCs) and is downregulated upon differentiation, suggesting a potential role for Matn4 in HSCs. While constitutive Matn4 KO mice exhibit normal hematopoiesis under homeostasis, their HSCs show a striking long-term growth advantage in competitive transplantation settings, outcompeting wt engraftment within 4 weeks after the transplantation. Confirming this, Matn4 KO LSK cells show higher colony forming and serial replating potential in vitro, which can be rescued by the addition of recombinant or overexpressed Matn4. Interestingly, Matn4 is downregulated in HSCs upon transplantation and other stress conditions such as IFNa treatment and in vitro culture. In response to these stress stimuli, HSCs lacking Matn4 showed increased cell cycle activation which may be explained in part by altered CXCR4 signaling in Matn4 KO HSCs, as indicated by expression profiling data. Critically, Matn4 KO HSCs return to quiescence normally after stressinduced activation and transplantation, thereby preventing their exhaustion. In summary, we show that the extracellular matrix protein Matn4 is a novel component of the HSC niche, regulating the HSC stress response. Surprisingly, HSCs lacking this extracellular matrix protein show a higher HSC potential due to an accelerated response to stress. Our data suggest that high expression of Matn4 in LT-HSCs confers a resistance to stress stimuli. However, in situations of acute stress, such as infection or transplantation, this protection is rapidly lost to allow HSCs to efficiently replenish the blood system.
2004 - OVERCOMING QUIESCENCE BY TARGETING P21 (CDKN1A) SENSITIZES PRE-LEUKEMIC STEM CELLS TO CHEMOTHERAPY Cedric S. Tremblay, Jesslyn Saw, Sung K. Chiu, and David Curtis Monash University, Melbourne, Victoria, Australia T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy, with 1 out of 5 patients dying from refractory disease. Recent studies support the concept that cells responsible for relapse frequently develop from a small population of quiescent pre-leukemic stem cells (pre-LSCs) that give rise to clonal heterogeneity. Using the Lmo2 transgenic mouse model, we have shown previously that these pre-LSCs have long-term self-renewal potential and resistance to high dose radiation. To determine if quiescence is an important property of pre-LSCs, we first used the doxycycline inducible H2B-GFP transgenic mouse model to show the existence of rare (!1%) pre-LSCs that cycle less than once a month in Lmo2 transgenic mice. Importantly, radiation therapy lead to a 5-fold enrichment of these rare preLSCs, confirming that quiescence protects pre-LSCs from genotoxic stress. We then used mice lacking the cell cycle regulator Cdkn1a (p21) to address the importance of quiescence in therapeutic resistance. Absence of p21 had no effect on the formation of pre-LSCs but reduced the proportion of quiescent pre-LSCs in vivo. Importantly, pre-LSCs lacking p21 were sensitive to killing by radiation therapy and chemotherapy. Finally, we aged cohorts of mice to determine the role of p21 in the clonal evolution of pre-LSCs. Remarkably, absence of p21 strongly impaired the clonal selection of pre-LSCs and the subsequent development of T-ALL. Gene expression analysis revealed that absence of p21 was associated with reduced expression of quiescence and increased expression of T-cell differentiation genes in preLSCs. The restored differentiation profile in Lmo2 mice lacking p21 together with these gene expression profiles indicate that loss of p21 triggers differentiation of pre-LSCs. These results provide the most convincing in vivo evidence that p21 is required for the quiescence, maintenance, clonal evolution and therapeutic resistance of pre-LSCs. Targeting p21 and other regulators of quiescence may represent a promising new therapeutic strategy for improving cure rates in T-ALL.
2006 - CLEC-2 SIGNALING IS CRUCIAL FOR THE PRODUCTION OF THROMBOPOIETIN IN THE MEGAKARYOCYTE NICHE Ayako Nakamura-Ishizu and Toshio Suda National University of Singapore, Singapore, Singapore The bone marrow (BM) microenvironment (i.e. the niche) governs the integrity of hematopoietic stem cells (HSCs) through the production of various niche factors. Recently, mature megakaryocytes (Mk) in the BM were characterized for their production of niche factors and their presence in the BM determined HSCs to remain quiescent. We reported that thrombopoietin (TPO) is one cytokine produced in Mks and functions as a secretory niche factor to maintain HSCs. However, specific molecular pathways that govern the niche functions of Mks are largely unknown. Analysis of mice with specific deletion of platelet activation receptor C-type lectin like receptor-2 (CLEC-2) on Mk lineages (PF4Cre:CLEC-2floxed/floxed;Clec2MkD/D) exhibit a characteristic decline in HSC stem cell potential despite a chiefly intact BM Mk cell number. Although Clec2MkD/D mice exhibit a modest decrease in mature Mks in the metaphyseal region of the BM, Clec2MkD/D Mk progenitors matured without arrest in normal ploidy. HSCs in Clec2MkD/D show exit from cell cycle quiescence and decreased repopulation potential. When co-cultured with HSCs, Clec2MkD/D Mks failed to maintain LT-HSC populations in vitro. CLEC-2 depleted Mks expressed significantly low transcript levels of various niche factors including TPO. Knockdown of CLEC-2 downstream targets, Lcp2, Syk and Plcg2, in Mks using shRNA exhibited a decline in Thpo transcript levels. Despite presenting thrombocytopenia in the peripheral blood, CLEC-2 deficient Mks exhibited low production of TPO signifying a defective feedback regulation of TPO production in the absence of platelet and Mk CLEC-2. Loss of quiescence of HSCs was dependent on TPO as administration of recombinant TPO rescued the HSC phenotypes in Clec2MkD/D mice. Podoplanin, a receptor for CLEC-2, was highly expressed on osteoblasts within the BM and co-localized with CLEC-2 positive Mks. Taken together, CLEC-2 signaling is crucial for the production of TPO in BM Mks in order to maintain quiescent HSCs. Our data signifies that TPO production in the BM has a discrete mechanism apart from systemic TPO regulations; a mechanism through which local niche cells may interrelate for a delicate fluctuation of niche factor production.
S45
Short Talk Presentations 2003 - RAPID FORMATION OF B CELL LEUKEMIA IN MICE EXPRESSING THE PAX5-JAK2 FUSION PROTEIN Sabine Jurado, Daniela Kostanova, and Meinrad Busslinger Research Institute of Molecular Pathology, Vienna, Austria
2005 - EXTRACELLULAR MATRIX PROTEIN MATN4 REGULATES HSC STRESS RESPONSE Hannah Uckelmann, Sandra Blaszkiewicz, and Marieke Essers DKFZ/HI-STEM, Heidelberg, Germany
Abnormalities in B cell development, regulation and survival can lead to immunodeficiency, autoimmunity and leukemia or lymphoma formation. Pax5 is considered to be the guardian of B cell identity; it is therefore not surprising that about a third of all B-cell precursor acute lymphoplastic leukemias (BCP-ALL) have heterozygous PAX5 alterations, including gene rearrangements. Among them are translocations that lead to the expression of fusion proteins, which are thought to interfere with normal Pax5 function. However, heterozygous Pax5 mutations are not sufficient for tumor formation in mice. Hence, a second oncogenic hit is required, such as the constitutive activation of STAT5 or deletion of CDKN2a/b. One recently described in-frame fusion protein, PAX5-JAK2, results in the loss of the auto-inhibitory pseudo-kinase domain of JAK2, potentially leading to a constitutively active JAK2 kinase (caJAK2). caJAK2 could in turn promote constitutive activation of STAT5 and therefore provide the second oncogenic hit necessary for leukemia formation. In order to determine the oncogenic potential of PAX5-JAK2, we have generated a mouse expressing this fusion protein from the Pax5 locus. Here we show that heterozygous expression of the Pax5-JAK2 allele in these mice, mimicking the clinical situation, is sufficient to drive leukemia formation within 12 weeks. Flow cytometric analyses of leukemic cells reveal the presence of surface markers corresponding to early stages of B-cell development, similar to the BCP-ALL diagnosis of human patients. Abnormal B cells can already be detected accumulating in the bone marrow of 4-5 week-old mice, and are later found in secondary lymphoid organs. Interestingly, these tumor cells have higher phosphoSTAT5 levels than normal B cells and can also undergo loss of expression of the wild-type Pax5 allele. In conclusion, the fast and reproducible leukemia formation observed in these mice makes it a faithful model to study the initiation and maintenance of leukemia of Pax5-JAK2 positive BCP-ALL, and current work is aimed at unraveling the mechanism of action of Pax5-JAK2.
Multipotent hematopoietic stem cells (HSCs) are found in a dormant state for most of their lifetime, which they exit only rarely to self-renew or differentiate to sustain blood supply. In acute stress situations such as infection, HSCs are activated to reconstitute compromised blood cells. The molecular and cellular mechanisms controlling stress-induced activation of HSCs remain largely unknown. The extracellular matrix adaptor protein Matrilin-4 (Matn4) is highly expressed in long-term HSCs (LT-HSCs) and is downregulated upon differentiation, suggesting a potential role for Matn4 in HSCs. While constitutive Matn4 KO mice exhibit normal hematopoiesis under homeostasis, their HSCs show a striking long-term growth advantage in competitive transplantation settings, outcompeting wt engraftment within 4 weeks after the transplantation. Confirming this, Matn4 KO LSK cells show higher colony forming and serial replating potential in vitro, which can be rescued by the addition of recombinant or overexpressed Matn4. Interestingly, Matn4 is downregulated in HSCs upon transplantation and other stress conditions such as IFNa treatment and in vitro culture. In response to these stress stimuli, HSCs lacking Matn4 showed increased cell cycle activation which may be explained in part by altered CXCR4 signaling in Matn4 KO HSCs, as indicated by expression profiling data. Critically, Matn4 KO HSCs return to quiescence normally after stressinduced activation and transplantation, thereby preventing their exhaustion. In summary, we show that the extracellular matrix protein Matn4 is a novel component of the HSC niche, regulating the HSC stress response. Surprisingly, HSCs lacking this extracellular matrix protein show a higher HSC potential due to an accelerated response to stress. Our data suggest that high expression of Matn4 in LT-HSCs confers a resistance to stress stimuli. However, in situations of acute stress, such as infection or transplantation, this protection is rapidly lost to allow HSCs to efficiently replenish the blood system.
2004 - OVERCOMING QUIESCENCE BY TARGETING P21 (CDKN1A) SENSITIZES PRE-LEUKEMIC STEM CELLS TO CHEMOTHERAPY Cedric S. Tremblay, Jesslyn Saw, Sung K. Chiu, and David Curtis Monash University, Melbourne, Victoria, Australia T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy, with 1 out of 5 patients dying from refractory disease. Recent studies support the concept that cells responsible for relapse frequently develop from a small population of quiescent pre-leukemic stem cells (pre-LSCs) that give rise to clonal heterogeneity. Using the Lmo2 transgenic mouse model, we have shown previously that these pre-LSCs have long-term self-renewal potential and resistance to high dose radiation. To determine if quiescence is an important property of pre-LSCs, we first used the doxycycline inducible H2B-GFP transgenic mouse model to show the existence of rare (!1%) pre-LSCs that cycle less than once a month in Lmo2 transgenic mice. Importantly, radiation therapy lead to a 5-fold enrichment of these rare preLSCs, confirming that quiescence protects pre-LSCs from genotoxic stress. We then used mice lacking the cell cycle regulator Cdkn1a (p21) to address the importance of quiescence in therapeutic resistance. Absence of p21 had no effect on the formation of pre-LSCs but reduced the proportion of quiescent pre-LSCs in vivo. Importantly, pre-LSCs lacking p21 were sensitive to killing by radiation therapy and chemotherapy. Finally, we aged cohorts of mice to determine the role of p21 in the clonal evolution of pre-LSCs. Remarkably, absence of p21 strongly impaired the clonal selection of pre-LSCs and the subsequent development of T-ALL. Gene expression analysis revealed that absence of p21 was associated with reduced expression of quiescence and increased expression of T-cell differentiation genes in preLSCs. The restored differentiation profile in Lmo2 mice lacking p21 together with these gene expression profiles indicate that loss of p21 triggers differentiation of pre-LSCs. These results provide the most convincing in vivo evidence that p21 is required for the quiescence, maintenance, clonal evolution and therapeutic resistance of pre-LSCs. Targeting p21 and other regulators of quiescence may represent a promising new therapeutic strategy for improving cure rates in T-ALL.
2006 - CLEC-2 SIGNALING IS CRUCIAL FOR THE PRODUCTION OF THROMBOPOIETIN IN THE MEGAKARYOCYTE NICHE Ayako Nakamura-Ishizu and Toshio Suda National University of Singapore, Singapore, Singapore The bone marrow (BM) microenvironment (i.e. the niche) governs the integrity of hematopoietic stem cells (HSCs) through the production of various niche factors. Recently, mature megakaryocytes (Mk) in the BM were characterized for their production of niche factors and their presence in the BM determined HSCs to remain quiescent. We reported that thrombopoietin (TPO) is one cytokine produced in Mks and functions as a secretory niche factor to maintain HSCs. However, specific molecular pathways that govern the niche functions of Mks are largely unknown. Analysis of mice with specific deletion of platelet activation receptor C-type lectin like receptor-2 (CLEC-2) on Mk lineages (PF4Cre:CLEC-2floxed/floxed;Clec2MkD/D) exhibit a characteristic decline in HSC stem cell potential despite a chiefly intact BM Mk cell number. Although Clec2MkD/D mice exhibit a modest decrease in mature Mks in the metaphyseal region of the BM, Clec2MkD/D Mk progenitors matured without arrest in normal ploidy. HSCs in Clec2MkD/D show exit from cell cycle quiescence and decreased repopulation potential. When co-cultured with HSCs, Clec2MkD/D Mks failed to maintain LT-HSC populations in vitro. CLEC-2 depleted Mks expressed significantly low transcript levels of various niche factors including TPO. Knockdown of CLEC-2 downstream targets, Lcp2, Syk and Plcg2, in Mks using shRNA exhibited a decline in Thpo transcript levels. Despite presenting thrombocytopenia in the peripheral blood, CLEC-2 deficient Mks exhibited low production of TPO signifying a defective feedback regulation of TPO production in the absence of platelet and Mk CLEC-2. Loss of quiescence of HSCs was dependent on TPO as administration of recombinant TPO rescued the HSC phenotypes in Clec2MkD/D mice. Podoplanin, a receptor for CLEC-2, was highly expressed on osteoblasts within the BM and co-localized with CLEC-2 positive Mks. Taken together, CLEC-2 signaling is crucial for the production of TPO in BM Mks in order to maintain quiescent HSCs. Our data signifies that TPO production in the BM has a discrete mechanism apart from systemic TPO regulations; a mechanism through which local niche cells may interrelate for a delicate fluctuation of niche factor production.