ALBERT

Description: Oncogenic transcription factors are commonly activated in acute leukemias and subvert normal gene expression networks to reprogram hematopoietic progenitors into preleukemic stem cells, as exemplified by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). Whether or not these oncoproteins interfere with other DNA-dependent processes is largely unexplored. Here, we show that LMO2 is recruited to DNA replication origins by interaction with three essential replication enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic DNA sequences is sufficient to transform these sequences into origins of replication. We next addressed the importance of LMO2 in erythroid and thymocyte development, two lineages in which cell cycle and differentiation are tightly coordinated. Lowering LMO2 levels in erythroid progenitors delays G1-S progression and arrests erythropoietin-dependent cell growth while favoring terminal differentiation. Conversely, ectopic expression in thymocytes induces DNA replication and drives these cells into cell cycle, causing differentiation blockade. Our results define a novel role for LMO2 in directly promoting DNA synthesis and G1-S progression.

Description: Acute promyelocytic leukemia (APL) is a rare disease that is successfully treated with targeted therapy in the clinic using all-transretinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide (ATO). APL is invariably characterized by a translocation involving the retinoic acid receptor alpha (RARA) and genes encoding proteins with self-aggregation motifs. The translocation t(15;17) leads to the expression of the oncofusion protein PML-RARA that results in a differentiation arrest, an increase of self-renewal potential and an anti-apoptotic phenotype. Unfortunately, resistance to ATRA and ATO treatment are described in patients due to mutations in the RARA or PML moiety, respectively. Therefore, a better understanding of APL therapy induced molecular responses will allow to address treatment resistances. We and others described that current APL therapies induce autophagy. Autophagy is a proteolytic self-degradation process characterized by the formation of double-membraned vesicles, so called autophagosomes, which engulf cytoplasmic contents such as protein aggregates or defective organelles. In APL therapy autophagy is among others involved in PML-RARA degradation. We also found that key autophagy-related proteins such as WIPI1, DRAM1, ATG7 and ATG5 are essential for APL cells differentiation. A comprehensive study of APL therapy induced autophagy is needed to design meaningful combination therapies with autophagy modulating drugs. We previously found significantly lower Death-Associated Protein Kinase 2 (DAPK2) expression levels in acute myeloid leukemia (AML) patients with particular low levels in APL (Humbert et al, J Leukoc Biol 2014). DAPK2 is a positive mediator of autophagy and cell death in different cellular systems. To test if DAPK2 is also involved in autophagy and cell death responses to APL therapy, we first determined DAPK2 levels upon ATRA or ATO treatment. Both treatments significantly induced DAPK2 expression in NB4 and HT93 APL cell lines model paralleled by autophagy and cell death induction. Next, we used an immunoprecipitation screening approach to identify DAPK2 binding partners during APL therapy. We found that the known tumor suppressor p73 as well as the autophagy-related (ATG) protein ATG5 and a short form of Beclin1 that is involved in apoptosis co-precipitate with DAPK2. Then, we knocked down DAPK2 in NB4 cells via shRNAs and determined autophagic and cell death responses upon ATRA and ATO treatment. Autophagic flux was determined by endogenous Light Chain 3 (LC3)B-II puncta formation and levels by immunofluorescence microscopy and by western blot in presence or absence of the lysosomal inhibitor, Bafilomycin A1. Silencing of DAPK2 led to decreased autophagic activity upon ATRA treatment. In addition, DAPK2 depletion in NB4 cells caused a decrease in ATG5-ATG12 complex formation in both APL therapies as well as an accumulation of full-length Beclin1. Surprisingly, inhibiting DAPK2 expression did not significantly impair autophagic activity upon ATO treatment but attenuated ATO-induced apoptosis (p

Description: Abstract 3369 Intact DNA damage response pathways are important for genomic fidelity of cells in order to avoid tumor formation. On the other hand, inhibition of DNA repair provides an important mechanism to enhance the therapeutic efficacy of DNA damaging agents such as gamma-irradiation. Thus, it is important to identify novel players in DNA damage response that might represent novel targets for combination therapies. Death-associated protein kinases (DAPK) are serine/threonine kinases believed to be involved in cell death and autophagy mechanisms, whereby particularly the role of DAPK1 has previously been investigated. The DAPK family is composed of five members: DAPK1, DAPK2 (or DRP-1), DAPK3 (or ZIP kinase), DRAK1 and DRAK2. DAPK1 and DAPK2 share 80% homology in the catalytic domain. Generally, the role of DAPK in DNA damage responses is not well studied. To analyze the role of DAPK1 and DAPK2 in response to gamma-irradiation, we used p53 wild-type REH B-cell acute lymphoblastic leukemia (B-ALL) cells as a model. In response to irradiation, DAPK1 protein expression increased paralleled by an increased of total p53, phospho-Ser20-p53 and p21WAF1/CIP1. DAPK2 expression, however, did not increase. Since upregulation of p21WAF1/CIP1, a classical p53 target in response to DNA damage leads to cell cycle arrest, we asked whether knocking down DAPK1 or DAPK2 might affect the cell cycle. Interestingly, knocking down DAPK2 but not DAPK1 led to a significant increase of S-phase cells upon irradiation. Moreover, knocking down DAPK2 attenuated the induction of DAPK1 upon irradiation indicating a DAPK2-DAPK1 cascade in DNA damage responses. Next, given the significant role of p21WAF1/CIP1 and p53 in DNA damage responses, we tested if DAPK2 might directly participate in a novel signaling pathway by interacting with these proteins. Indeed, pull down assays revealed that p21WAF1/CIP1 and p53 are novel DAPK2 interacting proteins. Clearly, further experiments are needed to define the DAPK2-DAPK1-p53- p21WAF1/CIP1 network in DNA repair pathways. In conclusion, we identified a novel role for DAPK1 and DAPK2 in DNA damage responses of B-ALL cells and propose a novel DAPK2/DAPK1/p53/ p21WAF1/CIP1 DNA damage regulatory pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is characterized by a block in myeloid differentiation and an aberrant cell proliferation of leukemia blasts. While other AML therapies cure only 30% of the patients, acute promyelocytic leukemia (APL) is successfully treated by differentiation therapy using all-trans retinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide. A better understanding of the molecular mechanisms underlying ATRA therapy in APL may offer new perspectives in the treatment of additional AML subtypes. Fatty acid synthase (FASN) is the only human lipogenic enzyme able of de novo fatty acid synthesis. Its expression is very low in healthy adult tissues, whereas increased expression is commonly found in a variety of cancerous tissues. Therefore, FASN represents an attractive potential drug target. To investigate if a similar expression pattern is found in normal versus malignant myeloid cells, we determined FASN expression in fresh leukemic blast cells from untreated AML patients obtained at the Inselspital Bern (Switzerland) (n=68) and expression data available online from the Bloodspot server (n=203). In both AML cohorts, we found that FASN expression is significantly higher in AML patients than in healthy granulocytes (p

Description: Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein. We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively. To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity. Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation. Disclosures No relevant conflicts of interest to declare.

Description: The transcription factor PU.1 (SPI-1) plays an important role in numerous cellular processes of myeloid cells, such as cell survival, proliferation and differentiation. PU.1 is expressed at intermediate levels in hematopoietic progenitor cells, whereas progenitors expressing low amounts of PU.1 differentiate towards the lymphoid lineage, while increased amounts of PU.1 levels promoted macrophage or granulocyte differentiation. Additionally, low PU.1 expression levels contribute to the immature myeloid phenotype in acute myeloid leukemia (AML). We recently linked PU.1 expression to TRAIL and chemotherapy sensitivity in AML cells. To further molecularly dissect the functions of PU.1 in myeloid cells, we focused on putative PU.1 targets associated with cell death responses emerging from a gene expression profiling experiment using Pu.1-null and Pu.1-restored 503 murine myeloid cells. We identified Caspase 8 (Casp8) and its paralogous gene c-Flip (aka FADD-like apoptosis regulator; Cflar) as PU.1-regulated genes, and showed putative PU.1-binding sites in their proximal promoter regions. CASP8 and c-FLIP are known for their function downstream of death-receptor mediated apoptosis. Yet, it has been reported that CASP8 has non-apoptotic functions involving cell proliferation, differentiation, and inflammation. C-FLIP is the enzymatically inactive homolog of CASP8 and exists in three isoforms: a long isoform (c-FLIPL), that partially inhibits CASP8 activity, and two short forms (c-FLIPR and c-FLIPS) that are anti-apoptotic. To assess if CASP8 and/or c-FLIP are involved in PU.1-regulated cellular processes, we took advantage of the NB4 APL cell line model. These cells can be differentiated towards granulocytes with all-trans retinoic acid (ATRA) in a PU.1-dependent manner. We found an 8-10-fold induction of CASP8 and c-FLIP mRNA expression upon granulocytic differentiation of NB4 cells. Underlining a possible function of these two genes in granulocyte differentiation, we detected a markedly increased mRNA expression of both genes in human CD34+ hematopoietic cells differentiated towards neutrophils using G-CSF. Furthermore, knocking down PU.1 in NB4 cells significantly impaired CASP8 and cFLIP mRNA upregulation. Importantly, the anti-apoptotic cFLIPs isoform was exclusively induced after prolonged ATRA-treatment in PU.1 knockdown cells, whereas cFLIPL and CASP8 mRNA levels were reduced. The binding of PU.1 to the CFLAR promoter region together with altered cFLIP isoform ratio upon PU.1 expression indicates direct transcriptional activation of cFLIP and possibly an involvement of PU.1 in alternative splicing of cFLIP. Based on previous reports linking non-apoptotic CASP8 functions to macrophage differentiation and our findings of PU.1-dependent CASP8 regulation, we next studied the role of CASP8 in more detail during monocyte and macrophage differentiation by knocking down CASP8 expression in HL60 cells. HL60 CASP8 knockdown cells, generated using two independent shRNAs, were treated with vitamin D3 (VitD3) or PMA to induce monocyte or macrophage differentiation, respectively. Surprisingly, knocking down CASP8 led to increased CD11b expression together with increased pseudopodia formation. Furthermore, analysis of secreted cytokines in HL60 CASP8 knockdown cells suggests activation of macrophages towards an M2 phenotype. Our findings extend the role of PU.1 function to cell survival during granulocytic differentiation of APL cells. This occurs via distinct regulation of the pro-apoptotic CASP8, and anti-apoptotic cFLIP gene programs, respectively. Our findings suggest that increased expression of the anti-apoptotic, shorter cFLIP isoforms later in neutrophil differentiation may support short-term neutrophil cell survival. Lastly, our results implicate a novel PU.1-CASP8 pathway that may be necessary for alternative activation of M2 macrophages. Disclosures No relevant conflicts of interest to declare.

Description: Autophagy is an intracellular degradation system that ensures a dynamic recycling of cytoplasmic contents. Autophagy is required for self-renewal and cell survival under stress caused by a variety of stimuli including starvation and chemotherapy. There is accumulating evidence for additional functions of autophagy during myeloid development and hematopoietic stem cell maintenance. In this study, we used primary acute myeloid leukemia (AML) samples and human APL/AML cell lines to investigate the autophagy pathway active in all-trans retinoic acid (ATRA) mediated neutrophil differentiation. By characterizing the autophagic pathway during neutrophil differentiation of APL cells in more detail, we identified a non-canonical autophagy pathway, which not necessarily requires a hierarchal involvement of all autophagy-related (ATG) proteins. In addition to previous findings, from us and others, showing that ATRA-induced autophagy in APL cells is Beclin-1 independent, we discovered that ATRA-induced autophagy during APL differentiation is dependent on only one ATG16L isoform. The ATG16L proteins ATG16L1 and L2 are part of the ubiquitin-like conjugation systems ATG12-ATG5-ATG16L1 and ATG8/LC3 that are essential for phagophore elongation and autophagosome maturation. ATG16L2 is an isoform of ATG16L1, which is dispensable for starvation-induced autophagy despite forming an ATG12-ATG5-ATG16L2 complex in COS-7 cells. By investigating ATG16 gene expression in acute myeloid leukemia (AML) blast cells, we found that ATG16L1 as well as L2 are significantly downregulated in primary AML patient samples. In addition, neutrophil differentiation of APL/AML cell lines and CD34+ myeloid progenitor cells resulted in a significant induction of ATG16L1 and ATG16L2 expression. Induction of ATG16L2 was clearly more prominent than that of ATG16L1. Importantly, knocking down ATG16L2 but not ATG16L1 significantly attenuated neutrophil differentiation of AML cells as evidenced by decreased expression of the differentiation markers CD11b, GCSFR and CEBPE. Moreover, inhibition of ATG16L2 but not ATG16L1 resulted in decreased autophagy induction upon ATRA-treatment. Conversely, silencing ATG16L1 but not ATG16L2 was able to inhibit canonical starvation but not ATRA-induced differentiation associated autophagy in APL cells. Our data reveal distinct functions of ATG16L1 and ATG16L2 in starvation and ATRA-induced autophagy. To investigate the transcriptional regulation of ATG16L2 during neutrophil differentiation, we screened the ATG16L2 promoter region for putative transcription factor binding sites. We identified PU.1 as a transcriptional regulator of ATG16L2 using chromatin immunoprecipitation, PU.1 knockdown APL cells and a PU.1 inducible AML cell line model. These findings are in line with our earlier findings that PU.1 activates transcription of the ATG genes WIPI1, ATG3, MAP1S and ATG4C during APL differentiation. Our data provide strong evidence for a particular, non-canonical subtype of autophagy operative during neutrophil differentiation of APL cells. ATG16L2, in contrast to ATG16L1 is essential for successful ATRA-induced neutrophil differentiation and autophagy. This is in sharp contrast to its lack of function during starvation-induced autophagy. Deciphering the particular autophagy pathway active during APL differentiation is a prerequisite to develop novel differentiation therapies that are based on autophagy modulation. Since our findings have been validated in non-APL cells, activation of autophagy might support neutrophil differentiation of AML cells in a more general way. Disclosures No relevant conflicts of interest to declare.

Description: Oncogenic transcription factors are major drivers in acute leukemias. These oncogenes are believed to subvert normal cell identity via the establishment of gene expression programs that dictate cell differentiation and growth. The LMO2 oncogene, which is commonly activated in T-cell acute lymphoblastic leukemia (T-ALL), has a well-established function in transcription regulation. We and others previously demonstrated that LMO1 or LMO2 collaborate with the SCL transcription factor to activate a self-renewal program that converts non self-renewing progenitors into pre-leukemic stem cells. Here we demonstrate a non-transcriptional role of LMO2 in controlling cell fate by directly promoting DNA replication, a hitherto unrecognized mechanism that might also account for its oncogenic properties. To address the question whether LMO2 controls other functions via protein-protein interactions, we performed a proteome-wide screen for LMO2 interaction partners in Kit+ Lin- cells. In addition to known LMO2-interacting proteins such as LDB1 and to proteins associated with transcription, we unexpectedly identified new interactions with three essential DNA replication enzymes, namely minichromosome 6 (MCM6), DNA polymerase delta (POLD1) and DNA primase (PRIM1). First, we show that in Kit+ hematopoietic cells (TF-1), all components of the pre-replication complex co-immunoprecipitate with LMO2 but not with SCL, suggesting a novel SCL-independent function. Second, LMO2 is recruited to DNA replication origins in these cells together with MCM5. Third, tethering LMO2 to synthetic DNA sequences is sufficient to transform these into origins of replication. Indeed, we show by DNA capture that LMO2 fused to the DNA binding domain of GAL4 is sufficient to recruit DNA replication proteins to GAL4 binding sites on DNA. In vivo, this recruitment is sufficient to drive DNA replication in a manner which is dependent on the integrity of the GAL4 binding sites. These results provide unambiguous evidence for a role of LMO2 in directly controlling DNA replication. Cell cycle and cell differentiation are tightly coordinated during normal hematopoiesis, both during erythroid differentiation and during thymocyte development. We next addressed the functional importance of LMO2 in these two lineages. Erythroid cell differentiation proceeds through different stages from the CD71+Ter119- to the CD71-Ter119+. These stages are also distinguishable by morphological criteria. We observe that LMO2 protein levels directly correlate with the proportion of cells in S phase, i.e. both LMO2 levels and the proportions of cycling cells decrease with terminal erythroid differentiation. Strikingly, lowering LMO2 levels in fetal liver erythroid progenitors via shRNAs decreases the proportion of cells in S phase and arrests Epo-dependent cell growth. Despite a drastic decrease in the numbers of erythroid precursors, these cells differentiate readily to the CD71-Ter119+ stage. Therefore, LMO2 levels dictate cell fate in the erythroid lineage, by favoring DNA replication at the expense of terminal maturation. Conversely, ectopic expression in thymocytes induces DNA replication and drives cells into cell cycle, causing differentiation blockade. Our results define a novel role for the oncogenic transcription factor LMO2 in directly promoting DNA synthesis. To our knowledge, this is the first evidence for a non-transcriptional function of the LMO2 oncogene that drives cell cycle at the expense of differentiation, favouring progenitor cell expansion in the thymus, and causing T-ALL when ectopically expressed in the T lineage. We propose that the non-transcriptional control of DNA replication uncovered here for LMO2 may be a more common function of oncogenic transcription factors than previously appreciated. Disclosures No relevant conflicts of interest to declare.

Description: Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.