Search

Integrated Stress Response (ISR) facilitates cellular adaptation to variable environmental conditions by reprogramming cellular response. Activation of ISR was reported in neurological disorders and solid tumours, but its function in hematological malignancies remains largely unknown. Previously we showed that ISR is activated in chronic myeloid leukemia (CML) CD34+ cells, and its activity correlates with disease progression and imatinib resistance. Here we demonstrate that inhibition of ISR by small molecule ISRIB, but not by PERK inhibitor GSK2656157, restores sensitivity to imatinib and eliminates CM Blast Crisis (BC) D34+ resistant cells. We found that in Patient Derived Xenograft (PDX) mouse model bearing CD34+ imatinib/dasatinib-resistant CML blasts with PTPN11 gain-of-function mutation, combination of imatinib and ISRIB decreases leukemia engraftment. Furthermore, genes related to SGK3, RAS/RAF/MAPK, JAK2 and IFNγ pathways were downregulated upon combined treatment. Remarkably, we confirmed that ISRIB and imatinib combination decreases STAT5 phosphorylation and inhibits expression of STAT5-target genes responsible for proliferation, viability and stress response. Thus, our data point to a substantial effect of imatinib and ISRIB combination, that results in transcriptomic deregulation and eradication of imatinib-resistant cells. Our findings suggest such drug combination might improve therapeutic outcome of TKI-resistant leukemia patients exhibiting constitutive STAT5 activation.

SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.

SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.

Acetylation of histones and nonhistone proteins is a posttranslational modification which plays a major role in the regulation of intracellular processes involved in tumorigenesis. It was shown that different acetylation of proteins correlates with development of leukemia. It is proposed that histone acetyltransferases (HATs) are important novel drug targets for leukemia treatment, however data are still not consistent. Our previous data showed that a derivative of anacardic acid - small molecule MG153, which has been designed and synthesized to optimize the HAT inhibitory potency of anacardic acid, is a potent inhibitor of p300/CBP associated factor (PCAF) acetyltransferase. Here we ask whether inhibition of PCAF acetyltransferase with MG153 will show proapoptotic effects in cells expressing BCR-ABL, which show increased PCAF expression and are resistant to apoptosis. We found that inhibition of PCAF decreases proliferation and induces apoptosis, which correlates with loss of the mitochondrial membrane potential and DNA fragmentation. Importantly, cells expressing BCR-ABL are more sensitive to PCAF inhibition compared to parental cells without BCR-ABL. Moreover, inhibition of PCAF in BCR-ABL-expressing cells breaks their resistance to DNA damage-induced cell death. These findings provide direct evidence that targeting the PCAF alone or in combination with DNA-damaging drugs shows cytotoxic effects and should be considered as a prospective therapeutic strategy in chronic myeloid leukemia cells. Moreover, we propose that anacardic acid derivative MG153 is a valuable agent and further studies validating its therapeutic relevance should be performed.

Activation of adaptive mechanisms plays a crucial role in cancer progression and drug resistance by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Our previous work showed that ER stress is induced in chronic myeloid leukemia (CML) cells. Herein, we demonstrate that the PERK-eIF2α phosphorylation pathway is upregulated in CML cell lines and CD34+ cells from CML patients and is associated with CML progression and imatinib resistance. We also show that induction of apoptosis by imatinib results in the downregulation of the PERK-eIF2α phosphorylation arm. Furthermore, we demonstrate that inactivation of the PERK-eIF2α phosphorylation arm decreases the clonogenic and proliferative capacities of CML cells and sensitizes them to death by imatinib. These findings provide evidence for a pro-survival role of PERK-eIF2α phosphorylation arm that contributes to CML progression and development of imatinib resistance. Thus, the PERK-eIF2α phosphorylation arm may represent a suitable target for therapeutic intervention for CML disease.

Meeting abstracts Enhancing immune responses with immune-modulatory monoclonal antibodies (mAbs) directed to inhibitory immune-receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as CTLA-4 or PD-1/

Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway in apoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), is involved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines from NBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin, however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed by cell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells only underwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from all three cell lines were able to activate the DDR pathway after being exposed to γ-radiation. Downregulation of nibrin in normal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence. Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damage dependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severely impaired, thus preventing the induction of senescence.

BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3'UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins - HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3'UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 -positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in "BRCAness" CML and BCR-ABL1 -positive ALL cells.

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53 mutations does not exclude the possibility that other dysfunctions play an important role in CML pathology. Acetylation represents a very potent posttranslational mechanism regulating p53 stability, transcriptional activity and localization. In this study we have investigated whether the expression of BCR-ABL could influence the acetylation of p53, specifically at lysine 317/320 (K317/K320), which has been shown to regulate nuclear export and transcription-independent apoptotic activity of p53. We found that BCR-ABL expression increases K317 acetylation of p53 and is able to prevent a drop in acetylation observed upon DNA damage, followed by translocation of p53 to the cytoplasm and by Bax activation. We have shown that this site plays a crucial role in the regulation of p53 localization and p53-dependent, Bax-mediated apoptosis. Our study presents a novel BCR-ABL-dependent mechanism protecting from DNA-damage-induced cell death. It can, in addition to already known mechanisms, explain the resistance to p53-dependent apoptosis observed in CML cells expressing wt p53. We propose that the acetyltransferases regulating the p53 acetylation could be an interesting and potent target for therapeutic intervention.

1.1 Genomic and chromosomal instability in CML An unstable genome is a common hallmark of nearly all solid tumors and most of leukemias in contrast to normal, healthy cells which are able to maintein genome integrity (Negrini et al., 2010). Genomic instability could result from changes in chromosome structure and number as well as changes on the DNA level. Chromosomal instability (CIN) arises from unproper chromosome segregation as well as division defects and leads to aneuploidy (Foijer, 2010), whereas accumulation of mutations and DNA alterations usually is an effect of the defective repair systems and DNA damage response in cancer cells (Economopoulou et al., 2011). Chronic myeloid leukemia (CML) cells expressing the BCR-ABL tyrosine kinase have been found to accumulate mutations as well as chromosomal abnormalities. One of the first indications that CML correlates with additional chromosome changes has been presented in 1987 (Alimena et al., 1987). Moreover, authors showed that the rate of chromosomal anomalies increased during the blastic transformation. In the next years this has been also confirmed by other authors (Hagemeijer, 1987; Johansson et al., 2002; Su et al., 1999; Suzukawa et al., 1997). Later, random aneuploidy rate between chromosomes 9 and 18 has been reported in CML patients – both, untreated as well as upon imatinib therapy (Amiel et al., 2006). In broader analysis of CML patients it was found that chromosomal instability caused by centrosomal aberrations significantly correlated with the disease progression (Giehl et al., 2005). In the chronic phase only one sample out of 18 showed additional karyotypic alterations, in contrast to blast crisis where 73% patients (11/16) displayed additional karyotype alterations. The observation that CML patients have karyotype aberrations was confirmed in other studies where complex chromosomal rearrangements (CCR) were investigated (Babicka et al., 2006). By using cytogenetics, the FISH, and multicolor FISH (mFISH) methods, a very high level of the genomic instability at the chromosomal level, in cells obtained from chronic myeloid leukemia patients was observed. Altogether, it was shown that the aberrations associated with the progression of BCR-ABLpositive CML chronic phase to the aggressive blast crisis include additional chromosomes (Ph1, +8, +19), isochromosome 17q (associated with the loss of p53), reciprocal

Recent findings showed that BRCA1, in addition to its role in DNA damage response, acts as an upstream regulator of genes involved in the mitotic checkpoint regulation, thus protecting against promotion of aberrant divisions and aneuploidy. Moreover, there is also an indication that the BRCA1 protein is downregulated in chronic myeloid leukemia (CML) patients. We have investigated a possible functional relationship between BRCA1 and mitotic checkpoint competence in cells with the same genetic background expressing different levels of Bcr-Abl, an oncogene responsible for CML. Herein, we show that Bcr-Abl strongly downregulates the BRCA1 protein level, which is partially reversed on treatment with imatinib, an inhibitor of Bcr-Abl tyrosine kinase. Bcr-Abl leads to decreased expression of genes involved in the mitotic checkpoint activation—Mad2, Bub1, Bub3, and BubR1, resulting in mitosis perturbances, weakened mitotic checkpoint function, and mitotic slippage after nocodazole treatment. Furthermore, high Bcr-Abl–expressing cells showed also postmitotic checkpoint dysfunctions and inability to effectively arrest in the 4NG1 phase of the cell cycle, which was associated with limited p21 induction. These observations had significant biological consequences, as we found a high level of improper divisions, chromosomal missegregation, and generation of polyploid cells on mitotic checkpoint prolonged activation. Additionally, Bcr-Abl–expressing cells showed resistance to death activated by spindle defects, reversed by imatinib. Our study presents new facts and supports the hypothesis concerning the mutator nature of Bcr-Abl itself. The functional interaction between Bcr-Abl and mitosis dysfunctions, due to compromised mitotic checkpoints, may have important implications for the generation of aneuploidy and CML progression. Mol Cancer Ther; 9(5); 1328–38. ©2010 AACR.