Jürg Schwaller

PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers

The identification as cooperating targets of Proviral Integrations of Moloney virus in murine lymphomas suggested early on that PIM serine/threonine kinases play an important role in cancer biology. Whereas elevated levels of PIM1 and PIM2 were mostly found in hematologic malignancies and prostate cancer, increased PIM3 expression was observed in different solid tumors. PIM kinases are constitutively active and their activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including several cell cycle regulators and apoptosis mediators. PIM1 but not PIM2 seems also to mediate homing and migration of normal and malignant hematopoietic cells by regulating chemokine receptor surface expression. Knockdown experiments by RNA interference or dominant-negative acting mutants suggested that PIM kinases are important for maintenance of a transformed phenotype and therefore potential therapeutic targets. Determination of the protein structure facilitated identification of an increasing number of potent small molecule PIM kinase inhibitors with in vitro and in vivo anticancer activity. Ongoing efforts aim to identify isoform-specific PIM inhibitors that would not only help to dissect the kinase function but hopefully also provide targeted therapeutics. Here, we summarize the current knowledge about the role of PIM serine/threonine kinases for the pathogenesis and therapy of hematologic malignancies and solid cancers, and we highlight structural principles and recent progress on small molecule PIM kinase inhibitors that are on their way into first clinical trials.

Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.

7,8-Dichloro-1-oxo-β-carbolines as a Versatile Scaffold for the Development of Potent and Selective Kinase Inhibitors with Unusual Binding Modes

Development of both potent and selective kinase inhibitors is a challenging task in modern drug discovery. The innate promiscuity of kinase inhibitors largely results from ATP-mimetic binding to the kinase hinge region. We present a novel class of substituted 7,8-dichloro-1-oxo-β-carbolines based on the distinct structural features of the alkaloid bauerine C whose kinase inhibitory activity does not rely on canonical ATP-mimetic hinge interactions. Intriguingly, cocrystal structures revealed an unexpected inverted binding mode and the presence of halogen bonds with kinase backbone residues. The compounds exhibit excellent selectivity over a comprehensive panel of human protein kinases while inhibiting selected kinases such as the oncogenic PIM1 at low nanomolar concentrations. Together, our biochemical and structural data suggest that this scaffold may serve as a valuable template for the design and development of specific inhibitors of various kinases including the PIM family of kinases, CLKs, DAPK3 (ZIPK), BMP2K (BIKE), and others.

Expression of leukemia-associated Nup98 fusion proteins generates an aberrant nuclear envelope phenotype

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98s N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Normal and pathological erythropoiesis in adults: from gene regulation to targeted treatment concepts

Pathological erythropoiesis with consequent anemia is a leading cause of symptomatic morbidity in internal medicine. The etiologies of anemia are complex and include reactive as well as neoplastic conditions. Clonal expansion of erythroid cells in the bone marrow may result in peripheral erythrocytosis and polycythemia but can also result in anemia when clonal cells are dysplastic and have a maturation arrest that leads to apoptosis and hinders migration, a constellation typically seen in the myelodysplastic syndromes. Rarely, clonal expansion of immature erythroid blasts results in a clinical picture resembling erythroid leukemia. Although several mechanisms underlying normal and abnormal erythropoiesis and the pathogenesis of related disorders have been deciphered in recent years, little is known about specific markers and targets through which prognosis and therapy could be improved in anemic or polycythemic patients. In order to discuss new markers, targets and novel therapeutic approaches in erythroid disorders and the related pathologies, a workshop was organized in Vienna in April 2017. The outcomes of this workshop are summarized in this review, which includes a discussion of new diagnostic and prognostic markers, the updated WHO classification, and an overview of new drugs used to stimulate or to interfere with erythropoiesis in various neoplastic and reactive conditions. The use and usefulness of established and novel erythropoiesis-stimulating agents for various indications, including myelodysplastic syndromes and other neoplasms, are also discussed.

Abstract IA23: Leukemia development and the microenvironment

Myeloproliferative neoplasms (MPNs) are hematological malignancies originated by hematopoietic stem cells (HSCs) carrying mutations, most frequently in JAK2, CALR or MPL genes. These diseases, which cannot be cured in most cases, are characterized by overproduction of myeloid and erythroid-megakaryocytic lineages. However, it remains unclear how some mutations, that are frequently common in disparate MPNs, can cause different clinical outcomes. Moreover, current treatments are mostly symptomatic and are only capable of slowing down the disease, but generally they cannot eradicate it. This suggests that other factors critically contribute to MPN progression. Our research group has demonstrated specific alterations of the microenvironment of HSCs in MPN. We have shown that mutated HSCs can only expand and trigger disease after damaging bone marrow (BM) nerve terminals and mesenchymal stem cells (BMSCs), overcoming the control that this environment can exert over mutated HSC proliferation and migration. Therefore, preserving the HSC niche might be a complementary strategy to treat these diseases. This approach is being currently tested in a multicenter phase-II clinical trial (clinicaltrials.gov identifier NCT02311569). MPNs can be considered a pre-leukemic state since MPN patients have a higher risk of developing acute myeloid leukemia (AML), which is highly aggressive and exhibits poor response to chemotherapy. Work from other groups and our preliminary data suggest that acute leukemic cells can transform the BM microenvironment to promote leukemogenesis. We have studied the contribution of nestin+ BMSCs to the development of acute myeloid leukemia (AML) induced by the MLL-AF9 oncogene. For this purpose we have used a doxycycline-inducible rtTA;MLL-AF9 mouse strain, which closely mimics the human disease. Development of AML in rtTA;MLL-AF9 mice caused a significant reduction of stromal cells in the BM but, contrasting our previous findings in MPN, it did not seem to affect the number of BM nestin+ cells. To study the possible contribution of nestin+ cells to leukemia development, we partially depleted nestin+ cells by inducing diphtheria toxin expression in these cells using Nestin-creERT2;iDTR mice previously transplanted in a competitive fashion with preleukemic and normal BM cells. We have shown previously that nestin+ cell depletion using this strategy can accelerate MPN. In contrast, nestin+ cell depletion had the opposite effect in AML, since it selectively diminished leukemia burden in BM, spleen and blood, and reduced the number of primitive leukemic cells, without affecting normal residual hematopoiesis. Combined nestin+ cell depletion and standard chemotherapy further exaggerated the elimination of BM leukemic cells. Among primitive leukemic cells, MLL-AF9+ lin- ckitlow sca1- cells were most significantly expanded in leukemia and were most significantly decreased upon nestin+ cell ablation, an effect that seemed to be further enhanced by simultaneous chemotherapy. This was not associated with detectable changes in the cell cycle profile of leukemic progenitors. Moreover, non-proliferative (ki67-) lin- cells were not found enriched in the vicinity of nestin+ cells, and Cxcl12 deletion in nestin+ cells, which increased thrombocytosis in our previous MPN studies, did not affect AML progression. In order to identify changes in leukemic progenitors following Nes+ cell depletion, we compared the gene expression profiles of lin- ckitlow sca1- cells isolated from control and Nes+ cell-depleted, MLL-AF9 expressing mice. Next-generation sequencing assays identified downregulation of gene sets related to HSC self-renewal, protein synthesis and degradation, and mitochondrial respiration in leukemic progenitors obtained from Nes+ cell-depleted bone marrow. The data suggests that AML-AF9+ leukemic progenitors impose changes in Nes+ cells that promote leukemogenesis. Using a novel co-culture system, we are currently testing possible mediators of the pro-survival effect of BMSCs over leukemic progenitors. Citation Format: Abel Sanchez-Aguilera, Vaia Stavropoulou, Daniel Martin-Perez, Alexandar Tzankov, Jurg Schwaller, Simon Mendez-Ferrer. Leukemia development and the microenvironment. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA23.

Modeling ETV6-JAK2-induced leukemia: insights from the zebrafish

Cloning and functional characterization of a large number of chromosomal translocations revealed that aberrant activation of protein kinases is a key event for expansion of the malignant clone in chronic myeloproliferative disorders, as well as in acute leukemia. The best known example is t(9;22)(