Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

Mouse APOBEC3 interferes with autocatalytic cleavage of murine leukemia virus Pr180gag-pol precursor and inhibits Pr65gag processing.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3.

Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy.

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

A small molecule compound IX inhibits telomere and attenuates oncogenesis of drug-resistant leukemia cells.

Drug resistance is a common obstacle in leukemia treatment and failing to eradicate leukemia stem cells is the main cause of leukemia relapse. Previous studies have demonstrated that telomerase activity is associated with deregulated self-renewal of leukemia stem cells (LSCs). Here, we identified a novel compound IX, an imatinib derivative with a replacement fragment of a telomerase inhibitor, which can effectively eradicate LSCs but had no influence on normal hematopoietic stem cells (HSCs) survival. We showed that compound IX can decrease the viability of drug-resistant K562/G cells and blast crisis CML primary patient cells. Besides, IX can affect LSC survival, inhibit the colony-forming ability, and reduce LSC frequency. In vivo results showed that IX can relieve the tumor burden in patient-derived xenograft (PDX) model and prolong the lifespan. We observed that compound IX can not only decrease telomerase activity, but also affect the alternative lengthening of telomeres. In addition, IX can inhibit both the canonical and non-canonical Wnt pathways. Our data suggested this novel compound IX as a promising candidate for drug-resistant leukemia therapy.

Aldophosphamide-thiazolidine (NSC-613060) an oxazaphosphorine cytostatic that crosses the blood brain barrier.

The pharmacologically active metabolite of cyclophosphamide is aldophosphamide. With cysteine, aldophosphamide forms stable aldophosphamide-thiazolidine which under physiological pH and temperature conditions hydrolyzes to aldophosphamide and cysteine. Aldophosphamide-thiazolidine was synthesized and tested for its ability as a cytostatic. The LD50 after a single intraperitoneal injection in mice was determined to be 2162 mg/kg, but after intravenous bolus administration of 500 mg/kg or in chronic toxicity tests with daily intraperitoneal injections, neurological side effects were observed. Antitumor activity was determined in therapy experiments in CD2F1 mice bearing subcutaneously transplanted P388 mouse leukemia cells. Administration of 100 mg/kg (less than 5% LD50) days 1-5 after tumor transplantation yielded an ILS of 100%. Organ distribution studies showed that aldophosphamide-thiazolidine is evenly distributed in all tissues examined, including brain tissue. The possibilities to increase the antitumor activity of aldophosphamide-thiazolidine by modulating the alkylating function are discussed.

Absence of the Shb gene in mixed-lineage leukemia MLL-AF9 cells increases latency in mice despite higher proliferation rates in vitro.

Mixed lineage leukemia (MLL) arises from several KMT2A-gene chromosomal translocations. Shb gene deficiency has been found to exhibit pleiotropic effects in different models of leukemia, and consequently, this study aimed to investigate MLL-AF9-induced leukemia in Shb deficiency. Bone marrow cells from wild type and Shb knockout (KO) mice were transduced with the MLL-AF9 gene. Shb KO MLL-AF9 cells proliferated at an increased rate, exhibited altered expression of certain cytokine genes (Kitl, Csf3, IL6, IL1b) and higher expression of cell cycle genes (Ccnd2, Ccne1). Mice receiving Shb KO MLL-AF9 cells showed longer latency without displaying any difference in rates of leukemic cell proliferation, indicating a dichotomy between the in vitro and in vivo phenotypes. The mice with Shb deficient MLL-AF9 cells had a lower content of leukemic bone marrow cells allowing elevated normal hematopoiesis, explaining the longer latency. Finally, Shb knockout GFP-positive bone marrow cells showed a higher percentage of cells expressing myeloid markers. The result suggests a role of Shb in the progression of leukemia and that the relevance of the Shb gene is context-dependent as inferred from the differences between the in vivo and in vitro responses. These findings help to obtain an increased understanding of human MLL-AF9 leukemia.

CD47 agonist peptide PKHB1 induces immunogenic cell death in T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.

Novel ETV6-RUNX1 Mouse Model to Study the Role of ELF-MF in Childhood B-Acute Lymphoblastic Leukemia: a Pilot Study.

Exposure to extremely low-frequency magnetic fields (ELF-MFs) has been classified by the International Agency for Research on Cancer (IARC) as "possibly carcinogenic to humans," based on limited scientific evidence concerning childhood leukemia. This assessment emphasized the lack of appropriate animal models recapitulating the natural history of this disease. Childhood B-cell acute lymphoblastic leukemia (B-ALL) is the result of complex interactions between genetic susceptibility and exposure to exogenous agents. The most common chromosomal alteration is the ETV6-RUNX1 fusion gene, which confers a low risk of developing the malignancy by originating a preleukemic clone requiring secondary hits for full-blown disease to appear. To develop potential prophylactic interventions, we need to identify the environmental triggers of the second hit. Recently, we generated a B-ALL mouse model of the human ETV6-RUNX1+ preleukemic state. Here, we present the results from the ARIMMORA pilot study, obtained by exposing 34 Sca1-ETV6-RUNX1 mice (vs. 27 unexposed) to a 50 Hz magnetic field of 1.5 mT with both fundamental and harmonic content, with an on/off cycle of 10 min/5 min, for 20 h/day, from conception until 3 months of age. Mice were monitored until 2 years of age and peripheral blood was periodically analyzed by flow cytometry. One of the exposed mice developed B-ALL while none of the non-exposed did. Although the results are statistically non-significant due to the limited number of mice used in this pilot experiment, overall, the results show that the newly developed Sca1-ETV6-RUNX1 mouse can be successfully used for ELF-MF exposure studies about the etiology of childhood B-ALL. Bioelectromagnetics. 2019;40:343-353. © 2019 Bioelectromagnetics Society.

Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1-/- mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1ß, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation.

CBFß-SMMHC creates aberrant megakaryocyte-erythroid progenitors prone to leukemia initiation in mice.

Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFß-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.

Cytoprotective autophagy maintains leukemia-initiating cells in murine myeloid leukemia.

Despite advances in the treatment of acute myeloid leukemia (AML), relapse and drug resistance frequently occur. Therefore, detailed mechanisms of refractoriness, including leukemia-initiating cell (LIC) biology, should be elucidated to treat AML. The self-degradative property of cytosolic macromolecules is central to autophagy and can contribute to homeostasis and stress response. Recent reports suggest the importance of autophagy in hematopoietic stem cells and various tumors. Thus, this study investigated the functional role of autophagy in AML maintenance and drug resistance using tamoxifen-inducible conditional knockout mice of Atg5 or Atg7, which are essential genes for autophagy, combined with an mixed lineage leukemia-eleven nineteen leukemia-induced murine AML model. Inactivation of autophagy by deletion of Atg5 or Atg7 prolonged survival in leukemic mice and reduced functional LICs. Atg7-deficient LICs displayed enhanced mitochondrial activity and reactive oxygen species production together with increased cell death. In addition, Atg7 deletion markedly decreased peripheral blood leukemia cells, concurrent with increased apoptosis, suggesting a higher dependency on autophagy compared with bone marrow leukemia cells. Finally, cytarabine (AraC) treatment activated autophagy in LICs, and Atg7 deletion potentiated the therapeutic effects of AraC, which included decreased LICs and prolonged survival, suggesting that autophagy contributes to AraC resistance. Our results highlight the intratumoral heterogeneity related to autophagy in AML and the unique role of autophagy in leukemia development and drug resistance.

NF-κB activation in chronic lymphocytic leukemia: A point of convergence of external triggers and intrinsic lesions.

The nuclear factor-κB (NF-κB) pathway is constitutively activated in chronic lymphocytic leukemia (CLL) patients, and hence plays a major role in disease development and evolution. In contrast to many other mature B-cell lymphomas, only a few recurrently mutated genes involved in canonical or non-canonical NF-κB activation have been identified in CLL (i.e. BIRC3, MYD88 and NFKBIE mutations) and often at a low frequency. On the other hand, CLL B cells seem 'addicted' to the tumor microenvironment for their survival and proliferation, which is primarily mediated by interaction through a number of cell surface receptors, e.g. the B-cell receptor (BcR), Toll-like receptors and CD40, that in turn activate downstream NF-κB. The importance of cell-extrinsic triggering for CLL pathophysiology was recently also highlighted by the clinical efficacy of novel drugs targeting microenvironmental interactions through the inhibition of BcR signaling. In other words, CLL can be considered a prototype disease for studying the intricate interplay between external triggers and intrinsic aberrations and their combined impact on disease evolution. In this review, we will discuss the current understanding of mechanisms underlying NF-κB deregulation in CLL, including micro-environmental, genetic and epigenetic events, and summarize data generated in murine models resembling human CLL. Finally, we will also discuss different strategies undertaken to intervene with the NF-κB pathway and its upstream mediators.

B Cell Linker Protein (BLNK) Is a Selective Target of Repression by PAX5-PML Protein in the Differentiation Block That Leads to the Development of Acute Lymphoblastic Leukemia.

PAX5 is a transcription factor that is required for the development and maintenance of B cells. Promyelocytic leukemia (PML) is a tumor suppressor and proapoptotic factor. The fusion gene PAX5-PML has been identified in acute lymphoblastic leukemia with chromosomal translocation t(9;15)(p13;q24). We have reported previously that PAX5-PML dominant-negatively inhibited PAX5 transcriptional activity and impaired PML function by disrupting PML nuclear bodies (NBs). Here we demonstrated the leukemogenicity of PAX5-PML by introducing it into normal mouse pro-B cells. Arrest of differentiation was observed in PAX5-PML-introduced pro-B cells, resulting in the development of acute lymphoblastic leukemia after a long latency in mice. Among the transactivation targets of PAX5, B cell linker protein (BLNK) was repressed selectively in leukemia cells, and enforced BLNK expression abrogated the differentiation block and survival induced by PAX5-PML, indicating the importance of BLNK repression for the formation of preleukemic state. We also showed that PML NBs were intact in leukemia cells and attributed this to the low expression of PAX5-PML, indicating that the disruption of PML NBs was not required for the PAX5-PML-induced onset of leukemia. These results provide novel insights into the molecular mechanisms underlying the onset of leukemia by PAX5 mutations.

Granulocytic myeloid-derived suppressor cells suppress virus-specific CD8+ T cell responses during acute Friend retrovirus infection.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) can suppress T cell responses in several different diseases. Previously these suppressive cells were observed to expand in HIV patients and in a mouse retrovirus model, yet their suppressive effect on virus-specific CD8+ T cells in vitro and in vivo has not been characterized thus far. RESULTS: We used the Friend retrovirus (FV) model to demonstrate that MDSCs expand and become activated during the late phase of acute FV infection. Only the subpopulation of granulocytic MDSCs (gMDSCs) but not monocytic MDSC suppressed virus-specific CD8+ T cell proliferation and function in vitro. gMDSCs expressed arginase 1, high levels of the inhibitory ligand PD-L1 and the ATP dephosphorylating enzyme CD39 on the cell surface upon infection. All three molecules were involved in the suppressive effect of the gMDSCs in vitro. MDSC depletion experiments in FV-infected mice revealed that they restrict virus-specific CD8+ T cell responses and thus affect the immune control of chronic retroviruses in vivo. CONCLUSIONS: Our study demonstrates that MDSCs become activated and expand during the acute phase of retrovirus infection. Their suppressive activity on virus-specific CD8+ T cells may contribute to T cell dysfunction and the development of chronic infection.

Ffar2 expression regulates leukaemic cell growth in vivo.

BACKGROUND: Activation of free fatty acid receptor 2 (FFAR2) by microbiota-derived metabolites (e.g., propionate) reduces leukaemic cell proliferation in vitro. This study aims to test whether Ffar2 expression per se also influences leukaemia cell growth in vivo. METHODS: Bcr-Abl-expressing BaF cells were used as a leukaemia model and the role of Ffar2 was evaluated in Balb/c mice after lentiviral shRNA transduction. RESULTS: Our data formally establish that reduced leukaemic cell proliferation is associated with increased Ffar2 expression in vivo and in vitro. Going beyond association, we point out that decreasing Ffar2 expression fosters cancer cell growth in vitro and in vivo. CONCLUSIONS: Our data demonstrate the role of Ffar2 in the control of leukaemic cell proliferation in vivo and indicate that a modulation of Ffar2 expression through nutritional tools or pharmacological agents may constitute an attractive therapeutic approach to tackle leukaemia progression in humans.

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection.

UNLABELLED: During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE: Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm-a crowded environment where diffusion is slow-is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.

Leukemic marrow infiltration reveals a novel role for Egr3 as a potent inhibitor of normal hematopoietic stem cell proliferation.

Cytopenias resulting from the impaired generation of normal blood cells from hematopoietic precursors are important contributors to morbidity and mortality in patients with leukemia. However, the process by which normal hematopoietic cells are overtaken by emerging leukemia cells and how different subsets of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are distinctly influenced during leukemic cell infiltration is poorly understood. To investigate these important questions, we used a robust nonirradiated mouse model of human MLL-AF9 leukemia to examine the suppression of HSCs and HPCs during leukemia cell expansion in vivo. Among all the hematopoietic subsets, long-term repopulating HSCs were the least reduced, whereas megakaryocytic-erythroid progenitors were the most significantly suppressed. Notably, nearly all of the HSCs were forced into a noncycling state in leukemic marrow at late stages, but their reconstitution potential appeared to be intact upon transplantation into nonleukemic hosts. Gene expression profiling and further functional validation revealed that Egr3 was a strong limiting factor for the proliferative potential of HSCs. Therefore, this study provides not only a molecular basis for the more tightened quiescence of HSCs in leukemia, but also a novel approach for defining functional regulators of HSCs in disease.

NrasG12D oncoprotein inhibits apoptosis of preleukemic cells expressing Cbfß-SMMHC via activation of MEK/ERK axis.

Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFß-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFß-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used Nras(LSL-G12D); Cbfb(56M) knock-in mice to show that allelic expression of oncogenic Nras(G12D) and Cbfß-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. Nras(G12D) and Cbfß-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfß-SMMHC. Furthermore, Nras(LSL-G12D); Cbfb(56M) leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfß-SMMHC and Nras(G12D) promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define Nras(LSL-G12D); Cbfb(56M) mice as a valuable genetic model for the study of inversion(16) AML-targeted therapies.

A novel colchicine-based microtubule inhibitor exhibits potent antitumor activity by inducing mitochondrial mediated apoptosis in MIA PaCa-2 pancreatic cancer cells.

Colchicine, an antimitotic alkaloid isolated from Colchicum autumnale, is a classical drug for treatment of gout and familial Mediterranean fever. It causes antiproliferative effects through the inhibition of microtubule formation, which leads to mitotic arrest and cell death by apoptosis. Here, we report that a novel colchicine analog, 4o (N-[(7S)-1,2,3-trimethoxy-9-oxo-10-[3-(trifluoromethyl)-4-chlorophenylamino]-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide), which exhibited potent anticancer activities both in vitro and in vivo. In this study, 4o with excellent pharmacokinetic profile and no P-gp induction liability displayed strong inhibition of proliferation against various human cancer cell lines. However, pancreatic cancer cell line MIA PaCa-2 was found to be more sensitive towards 4o and showed strong inhibition in concentration and time-dependent manner. By increasing intracellular reactive oxygen species (ROS) levels, 4o induced endoplasmic reticular stress and mitochondrial dysfunction in MIA PaCa-2 cells. Blockage of ROS production reversed 4o-induced endoplasmic reticulum (ER) stress, calcium release, and cell death. More importantly, it revealed that increased ROS generation might be an effective strategy in treating human pancreatic cancer. Further 4o treatment induced mitotic arrest, altered the expression of cell cycle-associated proteins, and disrupted the microtubules in MIA PaCa-2 cells. 4o treatment caused loss of mitochondrial membrane potential, cytochrome c release, upregulation of Bax, downregulation of Bcl-2, and cleavage of caspase-3, thereby showing activation of mitochondrial mediated apoptosis. The in vivo anticancer activity of the compound was studied using sarcoma-180 (ascitic) and leukemia (P388 lymphocytic and L1210 lymphoid) models in mice and showed promising antitumor activity with the least toxicity unlike colchicine. Such studies have hitherto not been reported. Taken together, these findings highlighted that 4o, a potent derivative of colchicine, causes tumor regression with reduced toxicity and provides a novel anticancer candidate for the therapeutic use.

CD150(-) Side Population Defines Leukemia Stem Cells in a BALB/c Mouse Model of CML and Is Depleted by Genetic Loss of SIRT1.

Leukemia stem cells (LSCs) of chronic myeloid leukemia (CML) are refractory to tyrosine kinase inhibitor treatment, persist in the residual disease, and are important source for disease recurrence. Better understanding CML LSCs will help devise new strategies to eradicate these cells. The BALB/c mouse model of CML using retroviral bone marrow transduction and transplantation is a widely used mouse model system for CML, but LSCs in this model are poorly characterized. Here, we show that lineage negative CD150(-) side population (CD150(-)SP), but not CD150(+)SP, are CML LSCs in this model, although both CD150(-)SP and CD150(+)SP cells are enriched for long-term hematopoietic stem cells in normal BALB/c mice. We previously showed that BCR-ABL transformation activates protein lysine deacetylase SIRT1 and inhibition of SIRT1 sensitizes CML stem/progenitor cells to tyrosine kinase inhibitors by acetylating and activating p53. In this study, we demonstrate that SIRT1 homozygous knockout substantially reduces CD150(-)SP CML LSCs, and compromises the maintenance of CML LSCs in the BALB/c model. We identified several molecular alterations in CD150(-)SP LSCs that included the elevated expression of cyclin-dependent kinase Cdk6 facilitating LSC activation and significantly reduced p53 expression. SIRT1 knockout suppressed Cdk6 expression and likely increases p53 protein functions through deacetylation without increasing its expression. Our results shed novel insight into CML LSCs and support a crucial role of SIRT1 in CML LSCs. Our study also provides a novel means for assessing new agents to eradicate CML LSCs.