Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.

The ribonucleoside AICAr induces differentiation of myeloid leukemia by activating the ATR/Chk1 via pyrimidine depletion.

Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage-associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.

Comparing the epidemiology, clinical characteristics and prognostic factors of acute myeloid leukemia with and without acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a particularly aggressive subtype of acute myeloid leukemia (AML), with high rates of early death. It is important to examine how epidemiological characteristics, clinical and treatment factors, cytogenetic and genetic data affect survival and differ between APL and non-APL AML patients. We analyzed population data from the New York State Cancer Registry to characterize AML including APL incidence rates by demographics. APL incidence rates were higher among Hispanics than non-Hispanics [incidence rate ratio = 1.22; 95% confidence interval (CI) = 1.02-1.43]; and among foreign-born than USA-born persons. APL incidence rates increased more rapidly through 1995-2014 than non-APL AML; and its frequency increased faster among foreign-born persons. In a hospital cohort of 390 AML patients, the risk of death was significantly higher among APL patients with FLT3-internal tandem duplications than those without [hazard ratio (HR) = 11.74; 95% CI = 1.03-134.5]; and among APL patients with secondary versus de novo disease (HR = 17.32; 95% CI = 1.56-192.1). Among non-APL AML patients, risk of death was significantly associated with prior chemotherapy with antitubulin agents after adjusting for age, gender and ethnicity (adjusted HR = 3.30; 95% CI = 1.49-7.32); and separately with older age, unfavorable cytogenetics and complex karyotype. This study highlights FLT3-internal tandem duplications as a prognostic factor in APL and proposes consideration of prior antitubulin therapy as a prognostic factor in non-APL AML.

The PML domain of PML-RARα blocks senescence to promote leukemia.

In most acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Although expression of the human PML fusion in mice promotes leukemia, its efficiency is rather low. Unexpectedly, we find that simply replacing the human PML fusion with its mouse counterpart results in a murine PML-RARα (mPR) hybrid protein that is transformed into a significantly more leukemogenic oncoprotein. Using this more potent isoform, we show that mPR promotes immortalization by preventing cellular senescence, impeding up-regulation of both the p21 and p19(ARF) cell-cycle regulators. This induction coincides with a loss of the cancer-associated ATRX/Daxx-histone H3.3 predisposition complex and suggests inhibition of senescence as a targetable mechanism in APL therapy.

Chemotherapy impedes in vitro microcirculation and promotes migration of leukemic cells with impact on metastasis.

Although most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis therapy. Surprisingly, emerging evidence suggests that certain anti-cancer drugs such as paclitaxel and doxorubicin can actually promote metastasis, but the mechanism(s) behind their pro-metastatic effects are still unclear. Here, we use a microfluidic microcirculation mimetic (MMM) platform which mimics the capillary constrictions of the pulmonary and peripheral microcirculation, to determine if in-vivo-like mechanical stimuli can evoke different responses from cells subjected to various cancer drugs. In particular, we show that leukemic cancer cells treated with doxorubicin and daunorubicin, commonly used anti-cancer drugs, have over 100% longer transit times through the device, compared to untreated leukemic cells. Such delays in the microcirculation are known to promote extravasation of cells, a key step in the metastatic cascade. Furthermore, we report a significant (p < 0.01) increase in the chemotactic migration of the doxorubicin treated leukemic cells. Both enhanced retention in the microcirculation and enhanced migration following chemotherapy, are pro-metastatic effects which can serve as new targets for anti-metastatic drugs.

Incorporation of arsenic trioxide in induction therapy improves survival of patients with newly diagnosed acute promyelocytic leukaemia.

OBJECTIVES: For patients with acute promyelocytic leukaemia (APL), negative reading of a promyelocytic leukaemia/retinoic acid receptor-alpha (PML-RARα) transcript after induction therapy correlates with a good prognosis. However, in the majority of patients given all-trans retinoic acid (ATRA)/anthracycline-based induction therapy, PML-RARα transcript remains even when haematologic complete remission is achieved. To facilitate maximal therapeutic efficacy for patients with APL, this study tested whether the addition of arsenic trioxide (ATO) would increase the rate of molecular complete remission after ATRA/anthracycline-based induction therapy. METHODS: Seventy-three patients with APL were induced with a regimen (designated 'AAA') consisting of ATO in combination with ATRA and daunorubicin. After this, a consolidation phase of daunorubicin-based chemotherapy and maintenance therapy with ATRA, ATO and methotrexate was administered. The noted outcomes were rates of complete remission, overall survival and disease-free survival. In addition, PML-RARα transcripts were monitored in 48 patients via RT-PCR. RESULTS: Rates of complete remission, overall survival and 5-yr disease-free survival were 95.89%, 94.52% and 96.28%, respectively. At the preconsolidation checkpoint, 68.75% (33/48) of patients had a negative reading for the PML-RARα fusion transcript. These outcomes were not influenced by mutations in FLT3 (fms-related tyrosine kinase 3) or other prognostic factors. CONCLUSIONS: The addition of ATO in the induction regimen was associated with an improved overall outcome for patients with de novo APL, with rather low relapse rate and better long-term survival.

Arsenic trioxide inhibits the Hedgehog pathway which is aberrantly activated in acute promyelocytic leukemia.

BACKGROUND/AIM: Dysregulated Hedgehog (Hh) signaling has been implicated in several human malignancies. Hh signaling inhibitors are predicted to have a minimal effect when the Smoothened receptor is mutated. Implications that Gli proteins are molecular targets of arsenic trioxide (ATO) action prompted us to investigate the expression of Hh signaling in acute promyelocytic leukemia (APL) and the influence of ATO on the Hh signaling pathway in APL. METHODS: Quantitative real-time reverse transcription polymerase chain reaction and Western blot were employed to analyze the expression of Hh pathway components and the influence of ATO on the Hh signaling pathway in APL. RESULTS: The expression of Hh pathway components was significantly upregulated in APL. In newly diagnosed APL patients, Gli2 expression was significantly positively correlated with Gli1 (R = 0.57, p < 0.001) and Smo (R = 0.56, p < 0.001) and the expression of Hh pathway components was significantly higher in the high WBC group (p < 0.05). ATO can significantly downregulate the expression of Hh pathway components in vitro and in vivo (p < 0.05). CONCLUSION: The Hh pathway is aberrantly activated in APL and associated with a bad prognostic factor. ATO can effectively inhibit the expression of the Hh pathway. The obtained data give the first clinical evidence for the application of ATO in tumors exhibiting an aberrantly activated Hh pathway.

Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor 1 (WT1) gene in acute promyelocytic leukemia.

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4 %) patients. At least one copy of the minor SNP rs16754 allele (WT1(AG) or WT1(GG)) was detected in 30 (29 %) patients. Six patients (6 %) were homozygous for the minor allele (WT1(GG)) and this genotype was associated with higher WT1 mRNA copies (p = 0.018). FLT3 mutations were found in 37 % of patients and correlated with high WT1 mRNA expression (p = 0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1(AA)) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.

The regulatory role of cell mechanics for migration of differentiating myeloid cells.

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytoskeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.

[Effect of arsenic trioxide and daunorubicin on procoagulant activity of acute promyelocytic leukemia cells].

OBJECTIVE: To explore the effect of arsenic trioxide (ATO) and daunorubicin (DNR) on phosphatidylserine (PS) exposure and related procoagulant activity of acute promyelocytic leukemia (APL) cells. METHODS: Mononuclear cell and neutrophil isolated from whole blood of 12 healthy volunteers were used as control group while APL cells obtained from 12 newly diagnosed APL patients at First Affiliated Hospital, Harbin Medical University from March 2007 to February 2009 were used as experimental group. APL cells were treated with 1 µmol/L ATO and 1 µmol/L DNR for 24 h. PS exposure of APL cells were measured by flow cytometry and confocal microscopy. And the related procoagulant activity was detected by the assays of coagulation time and coagulation factor formation. Lactadherin was used as a probe for PS exposure and anticoagulant on the cells of 12 APL patients. RESULTS: ATO induced a decrease of PS exposure on APL cells by flow cytometry and no staining with lactadherin was observed under confocal microscopy. However, DNR induced the significantly elevated PS exposure and staining green with a rim pattern on membrane of APL cells was obtained. Coagulation time was (180 ± 25) s and (220 ± 41) s before and after treatment with ATO, respectively (P < 0.05). The formation of coagulation factors decreased after treatment with ATO (P < 0.05). While coagulation time was (180 ± 25) s and (80 ± 20) s before and after treatment with DNR, respectively (P < 0.05). The formation of coagulation factors increased after treatment with DNR (all P < 0.05). Lactadherin inhibited the procoagulant activities of DNR-treated APL cells (all P < 0.05). CONCLUSIONS: Procoagulant activity is positively correlated with the exposed PS of APL cells. ATO and DNR inhibited and enhanced procoagulant activity with decreased and increased PS exposure, respectively.

The CEBPA gene is down-regulated in acute promyelocytic leukemia and its upstream promoter, but not the core promoter, is highly methylated.

Impairment of CCAAT Enhancer Binding Protein alpha (CEBPA) function is a common finding in acute myeloid leukemia; nevertheless, its relevance for acute promyelocytic leukemia pathogenesis is unclear. We analyzed the expression and assessed the methylation status of the core and upstream promoters of CEBPA in acute promyelocytic leukemia at diagnosis. Patients with acute promyelocytic leukemia (n = 18) presented lower levels of CEBPA expression compared to healthy controls (n = 5), but higher levels than those in acute myeloid leukemia with t(8;21) (n = 9) and with inv(16) (n = 5). Regarding the core promoter, we detected no methylation in 39 acute promyelocytic leukemia samples or in 8 samples from controls. In contrast, analysis of the upstream promoter showed methylation in 37 of 39 samples, with 17 patients showing methylation levels over 30%. Our results corroborate data obtained in animal models showing that CEBPA is down-regulated in acute promyelocytic leukemia stem cells and suggest that epigenetic mechanisms may be involved.

Metabolic-energy-dependent movement of PML bodies within the mammalian cell nucleus.

Promyelocytic leukaemia (PML) nuclear bodies are present in most mammalian cell nuclei. PML bodies are disrupted by PML retinoic acid receptor alpha (RAR alpha) oncoproteins in acute promyelocytic leukaemia. These bodies contain numerous proteins, including Sp100, SUMO-1, HAUSP(USP7), CBP and BLM, and they have been implicated in aspects of transcriptional regulation or as nuclear storage depots. Here, we show that three classes of PML nuclear bodies can be distinguished, on the basis of their dynamic properties in living cells. One class of PML bodies is particularly noteworthy in that it moves by a metabolic-energy-dependent mechanism. This represents the first example of metabolic-energy-dependent transport of a nuclear body within the mammalian cell nucleus.

Nuclear translocation of the 1,25D3-MARRS (membrane associated rapid response to steroids) receptor protein and NFkappaB in differentiating NB4 leukemia cells.

1,25 Dihydroxyvitamin D(3) (1,25D(3)) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D(3), which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D(3)-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NFkappaB). In unstimulated cells, 1,25D(3)-MARRS can be co-immunoprecipitated with antibodies directed at NFkappaB, and NFkappaB is co-precipitated when antibodies against 1,25D(3)-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D(3)-MARRS and NFkappaB begin translocating to the nucleus within minutes of co-stimulation with 1,25D(3) and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D(3)-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NFkappaB and other factors with which it may be interacting.

[Molecular mechanism of tetramethylpyrazine to induce human promyelocytic HL-60 leukemia cells differentiation].

OBJECTIVE: To study the molecular mechanism of tetramethylpyrazine to induce human promyelocytic HL-60 leukemia cells differentiation. METHOD: The cell proliferation was determined by MTT. The differentiation of the cells was detected by NBT reduction test. Cellular morphology was observed by Wright's staining. Cell cycle distribution and the distribution of CD11b, CD14 were detected by flow cytometry. Then RT-PCR and Western blot assay were employed to detect the expressions of c-myc, p27, CDK2 and cyclinE1 in HL-60 cells after exposure to TMP. RESULT: TMP inhibited the proliferation in a dose and time dependent manner. TMP at the concentration of 200 mg x L(-1) to 300 mg x L(-1) induced unterminal differentiation of HL-60 cell and synergistically blocked the cell cycle progression of HL-60 cells in G0/G1 phase. The expression of c-myc was down-regulated as well as the protein expression of cyclin E and CDK2, while the mRNA and protein expression of P27 were remarkably up-regulated. CONCLUSION: Small doses of TMP induces differentiation of HL-60 cells throughout the cell cyde, as detected by a slower rate of accumulation in G0/G1, possibly by regulating the expression and activity of G1/S phase-related molecules.

Isochromosome der(17)(q10)t(15;17) in acute promyelocytic leukemia resulting in an additional copy of the RARA-PML fusion gene: report of 4 cases and review of the literature.

Isochromosome of the long arm of the derivative chromosome 17, originating from the translocation t(15;17) [ider(17)(q10)t(15;17) or ider(17q)] in acute promyelocytic leukemia (APL), is a rare chromosome aberration which has been associated with a poor prognosis. In the present study, we report on 4 male APL patients with ider(17q) and review the clinical, cytogenetic and molecular characteristics of all previously reported APL patients with ider(17q) in order to clarify the clinical features and outcome of these patients. The data presented in this study demonstrated that ider(17q), which resulted in an extra RARA-PML fusion gene, was more frequent in males than females (male/female ratio of 2.12/1), was associated with a rather low initial white blood cell count and did not confer an adverse prognosis in APL patients treated with all-trans-retinoic acid and chemotherapy. The most frequent additional chromosome change to ider(17q) was trisomy 8. Ider(17q) was observed in all subtypes of the PML-RARA fusion gene, but the frequency of the bcr1 subtype was increased. Cases of overrepresentation of the RARA-PML fusion gene and ider(17q) cases may help in elucidating the role of RARA-PML in leukemogenesis.

Case report: An unusual presentation of acute promyelocytic leukemia in a middle aged female mimicking dengue infection.

RATIONALE: Acute promyelocytic leukemia (APL) is an uncommon subtype of acute myeloid leukemia (AML). M3v phenotype is a less common presentation of APL and these patients usually present with leukocytosis and abnormal promyelocytes that are characterized by sparse granulation and are less likely to have faggot cells with multiple Auer rods. Distinguishing M3v phenotype from acute febrile illness can be challenging as the diagnosis relies on examination of peripheral smear. PATIENT CONCERNS: Fifty-seven-year-old female who presented after recent trip to Dominican Republic for high grade fever and gum bleeding. She was exposed to patients with Dengue fever during her stay. At presentation, patient had leukocytosis, thrombocytopenia, and urinalysis showing bacteria and white cell. She was started on treatment for urinary tract infection. Patient remained febrile and thrombocytopenia worsened. On day 2, flow cytometry of the peripheral smear showed 43% medium sized blasts. Fluorescence in situ hybridization was positive for promyelocytic leukemia/retinoic acid receptor alpha. DIAGNOSES: The patient was diagnosed with APL. INTERVENTIONS: Patient was started on treatment with all-trans retinoic acid and arsenic trioxide along with supportive care OUTCOMES:: Patient had a favorable clinical response and her symptoms subsided. LESSONS: Flow cytometry of the peripheral smear is key to diagnosis of suspected APL. One must maintain high suspicion for this life-threatening condition as early diagnosis saves lives.

Pondering the puzzle of PML (promyelocytic leukemia) nuclear bodies: can we fit the pieces together using an RNA regulon?

The promyelocytic leukemia protein PML and its associated nuclear bodies are hot topics of investigation. This interest arises for multiple reasons including the tight link between the integrity of PML nuclear bodies and several disease states and the impact of the PML protein and PML nuclear bodies on proliferation, apoptosis and viral infection. Unfortunately, an understanding of the molecular underpinnings of PML nuclear body function remains elusive. Here, a general overview of the PML field is provided and is extended to discuss whether some of the basic tenets of "PML-ology" are still valid. For instance, recent findings suggest that some components of PML nuclear bodies form bodies in the absence of the PML protein. Also, a new model for PML nuclear body function is proposed which provides a unifying framework for its effects on diverse biochemical pathways such as Akt signaling and the p53-Mdm2 axis. In this model, the PML protein acts as an inhibitor of gene expression post-transcriptionally via inhibiting a network node in the eIF4E RNA regulon. An example is given for how the PML RNA regulon model provided the basis for the development of a new anti-cancer strategy being tested in the clinic.

Antibody therapy for acute myeloid leukemia.

Due to the high rate of relapse in younger patients and the overall poor outcome in older patients, novel therapies are needed for the treatment of acute myeloid leukemia (AML). Monoclonal antibodies have become an important treatment modality in cancer therapy. Gemtuzumab ozogamicin (GO), an anti-CD33 immunoconjugate, was approved by the US Food and Drug Administration (FDA) for the treatment of elderly patients with relapsed AML who are not candidates for standard chemotherapy. Single-agent GO and combinations with standard chemotherapeutics have been explored extensively in this disease. Hepatotoxicity and delayed myelosuppression have been dose-limiting. Its toxicity profile is reduced with decreased doses of GO and even by administering only a single infusion. In patients with acute promyelocytic leukemia (APL), the addition of GO can produce molecular remissions and is well tolerated. Targeted immunotherapy with GO for treatment of AML has produced remissions. In order to reduce toxicity and improve efficacy, its optimal dose and schedule and pairing with other standard chemotherapeutic agents need to be defined better in large clinical trials.