Irene Riz

G1/S transcriptional networks modulated by the HOX11/TLX1 oncogene of T-cell acute lymphoblastic leukemia.

The HOX11/TLX1 homeobox gene is aberrantly expressed in a subset of T-cell acute lymphoblastic leukemia (T-ALL). Here, we employed oligonucleotide microarrays to compare the expression profiles of the K3P and Sil leukemic cell lines originating from patients with HOX11+ T-ALL to that of Jurkat cells, which originated from a distinct subtype of T-ALL (TAL1+). To distinguish potential HOX11 target genes from those characteristic of the stage of HOX11 leukemic arrest, we also performed gene expression analysis on Jurkat cells, genetically engineered to express exogenous HOX11. The resulting HOX11 gene expression signature, which was validated for representative signaling pathways by transient transfection of reporter constructs, was characterized by elevated expression of transcriptional programs involved in cell proliferation, including those regulated by E2F, c-Myc and cAMP response element-binding protein. We subsequently showed that ectopic HOX11 expression resulted in hyperphosphorylation of the retinoblastoma protein (Rb), which correlated with inhibition of the major Rb serine/threonine phosphatase PP1. HOX11 also inhibited PP2A serine/threonine phosphatase activity concomitant with stimulation of the AKT/PKB signaling cascade. These results suggest that transcriptional deregulation of G1/S growth-control genes, mediated in large part through blockade of PP1/PP2A phosphatase activity, plays an important role in HOX11 pathobiology.

Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1

Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy‐resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P‐glycoprotein, a member of the ABC (ATP‐binding cassette) superfamily of transporters encoded by ABCB1. We also demonstrate that ABCB1‐overexpressing MM cells are resistant to the second‐generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy‐refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell‐surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1‐mediated drug resistance should it occur. Am. J. Hematol. 88:265–272, 2013.

TLX1/HOX11-induced hematopoietic differentiation blockade

Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.

Role of TLX1 in T-cell acute lymphoblastic leukaemia pathogenesis.

The diverged homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11 or TCL3) is activated in ∼5-10% of childhood and up to 30% of adult T-cell acute lymphoblastic leukemia (T-ALL) cases, most frequently by t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations which juxtapose the intact TLX1 coding region downstream of T cell receptor (TCR) δ (TRD@) or TCRβ (TCRB) gene regulatory sequences. TLX1+ T-ALL samples are virtually all arrested at the early cortical (CD1+) CD4+CD8+ “double-positive” (DP) stage of thymocyte development (Ferrando et al, 2002; Asnafi et al, 2004).

An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates

In this study, we utilized an integrated bioinformatics and computational biology approach in search of new BH3-only proteins belonging to the BCL2 family of apoptotic regulators. The BH3 (BCL2 homology 3) domain mediates specific binding interactions among various BCL2 family members. It is composed of an amphipathic α-helical region of approximately 13 residues that has only a few amino acids that are highly conserved across all members. Using a generalized motif, we performed a genome-wide search for novel BH3-containing proteins in the NCBI Consensus Coding Sequence (CCDS) database. In addition to known pro-apoptotic BH3-only proteins, 197 proteins were recovered that satisfied the search criteria. These were categorized according to α-helical content and predictive binding to BCL-xL (encoded by BCL2L1) and MCL-1, two representative anti-apoptotic BCL2 family members, using position-specific scoring matrix models. Notably, the list is enriched for proteins associated with autophagy as well as a broad spectrum of cellular stress responses such as endoplasmic reticulum stress, oxidative stress, antiviral defense, and the DNA damage response. Several potential novel BH3-containing proteins are highlighted. In particular, the analysis strongly suggests that the apoptosis inhibitor and DNA damage response regulator, AVEN, which was originally isolated as a BCL-xL-interacting protein, is a functional BH3-only protein representing a distinct subclass of BCL2 family members.

Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming

Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62. The eukaryotic translation initiation factor gene EIF4E3 was among the Nrf2 target genes upregulated in LP-1/Cfz cells, suggesting existence of a positive feedback loop. In line with this, we found that siRNA knockdown of eIF4E3 decreased Nrf2 protein levels. On the other hand, elevated SQSTM1/p62 levels were due at least in part to activation of the PERK-eIF2α pathway. LP-1/Cfz cells had decreased levels of reactive oxygen species as well as elevated levels of fatty acid oxidation and prosurvival autophagy. Genetic and pharmacologic inhibition of the Nrf2-EIF4E3 axis or the PERK-eIF2α pathway, disruption of redox homeostasis or inhibition of fatty acid oxidation or autophagy conferred sensitivity to carfilzomib. Our findings were supported by clinical data where increased EIF4E3 expression was predictive of Nrf2 target gene upregulation in a subgroup of patients with chemoresistant minimal residual disease and relapsed/refractory MM. Thus, our data offer a preclinical rationale for including inhibitors of the SQSTM1/p62-Nrf2 pathway to the treatment regimens for certain advanced stage MM patients.

Apoptotic Role of IKK in T-ALL Therapeutic Response

Despite considerable progress in the treatment of T cell acute lymphoblastic leukemia (T-ALL), it is still the highest risk malignancy among ALL. The outcome of relapsed patients remains dismal. The pro-survival role of NOTCH1 and NFκB in T-ALL is well documented; also, both factors were reported to be predictive of relapse. The NOTCH1 signaling pathway, commonly activated in T-ALL, was shown to enhance the transcriptional function of NFκB via several mechanisms. Thus, pharmacological inhibition of NOTCH1-NFκB signaling was suggested to be incorporated into existing T-ALL treatment protocols. However, conventional chemotherapy is based on activation of various types of stress, such as DNA damage, mitotic perturbations or endoplasmic reticulum overload. NFκB is frequently activated in response to stress and, depending on yet unknown mechanisms, it either protects cells from the drug action or mediates apoptosis. Here, we report that T-ALL cells respond to NFκB inhibition in opposite ways depending on whether they were treated with a stress-inducing chemotherapeutic agent or not. Moreover, we found that NOTCH1 enhances NFκB apoptotic function in the stressed cells. The data argue for further studies of NFκB status in T-ALL patients on different treatment protocols and the impact of activating NOTCH1 mutations on treatment response. Mol Cancer Res; 9(8); 979–84. ©2011 AACR.

Genomic Stability in Stem Cells

Accumulated data suggest that the unique function of stem cells to self-renew is under the strong control of a sensor system detecting potential threats to genomic integrity. Reactive oxygen species, the most significant mutagens in stem cells, when elevated, activate the protective mechanisms blocking self-renewal and at the same time serve as a signal stimulating stem cell differentiation. Based on studies performed primarily on hematopoietic stem cells and embryonic stem cells, we outline the signaling networks connecting the ATM, MAPK14 (p38) and FRAP1 (mTOR) protein kinases, the p53 tumor suppressor, the PTEN phosphatase, and the TEL, NF\(\upkappa\)B, FoxO, and HIF transcription factor families as a potential stress-controlled differentiation mechanism regulating stem cell fate decisions. An intriguing observation to come from these studies is that stem cell self-renewal capacity appears to be pharmacologically preserved by anti-oxidative agents.

Hematopoietic immortalizing function of the NKL-subclass homeobox gene TLX1.

Translocations resulting in ectopic expression of the TLX1 homeobox gene (previously known as HOX11) are recurrent events in human T‐cell acute lymphoblastic leukemia (T‐ALL). Transduction of primary murine hematopoietic stem/progenitor cells with retroviral vectors expressing TLX1 readily yields immortalized hematopoietic progenitor cell lines. Understanding the processes involved in TLX1‐mediated cellular immortalization should yield insights into the growth and differentiation pathways altered by TLX1 during the development of T‐ALL. In recent clinical gene therapy trials, hematopoietic clonal dominance or T‐ALL‐like diseases have occurred as a direct consequence of insertional activation of the EVI1, PRDM16 or LMO2 proto‐oncogenes by the retroviral vectors used to deliver the therapeutic genes. Additionally, the generation of murine hematopoietic progenitor cell lines due to retroviral integrations into Evi1 or Prdm16 has also been recently reported. Here, we determined by linker‐mediated nested polymerase chain reaction the integration sites in eight TLX1‐immortalized hematopoietic cell lines. Notably, no common integration site was observed among the cell lines. Moreover, no insertions into the Evi1 or Prdm16 genes were identified although insertion near Lmo2 was observed in one instance. However, neither Lmo2 nor any of the other genes examined surrounding the integration sites showed differential vector‐influenced expression compared to the cell lines lacking such insertions. While we cannot exclude the possibility that insertional side effects transiently provided a selective growth/survival advantage to the hematopoietic progenitor populations, our results unequivocally rule out insertions into Evi1 and Prdm16 as being integral to the TLX1‐initiated immortalization process.

p53 as a Therapeutic Target in T-ALL

TP53 is a central hub integrating stress signals from oncogenic genetic lesions and cytotoxic anti-cancer agents. The function of p53 as a regulator of transcription is well documented. More recently it was shown to directly interact with BCL2 family members and induce mitochondrial cell death. Stress-induced activation of p53 leads to cell cycle arrest that allows metabolic adjustments and repair mechanisms to take place prior to the next cycle; p53 may also induce senescence or apoptosis depending on the strength and nature of stress stimuli and/or cell type. In 50% of human cancers, the TP53 gene is deleted or mutated with a high proportion of gain of oncogenic function mutations. It is noteworthy therefore that TP53 is rarely mutated in T-ALL. However, its tumor suppressor activity is circumvented by genetic lesions. We will discuss here the most frequent T-ALL genetic abnormalities of INK4A/ARF, NOTCH1 and PTEN genes and how they affect TP53 expression and function. Current understanding of the signaling pathways governed by these oncogenes is advanced enough to find points of intersection with p53 downstream targets and attempt to translate the accumulated knowledge to the clinic. In addition, we will discuss the results of our analysis of publicly available expression profiling data indicating the existence of a TP53-anchored transcriptional program targeted by T-ALL oncogenes such as NOTCH, MYC and TLX1 in primary leukemic cells and how it can be exploited for cancer intervention. Overall, the goal of this chapter is to describe how T-ALL pathobiology affects the p53interacting network, highlighting some new potential therapeutic targets as well as some still unresolved questions.