Ethan Dmitrovsky

Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-Trans-retinoic acid)

BACKGROUND AND METHODS Patients with acute promyelocytic leukemia have a characteristic (15;17) translocation, with a breakpoint on chromosome 17 in the region of the retinoic acid receptor-alpha (RAR-alpha). Since this receptor has been shown to be involved with growth and differentiation of myeloid cells in vitro, and since recent clinical studies have reported that tretinoin (all-trans-retinoic acid) induces complete remission in patients with acute promyelocytic leukemia we studied the effects of tretinoin on cellular maturation and molecular abnormalities in patients undergoing the induction of remission with this agent. RESULTS Eleven patients with acute promyelocytic leukemia were treated with tretinoin administered orally at a dose of 45 mg per square meter of body-surface area per day. Nine of the 11 patients entered complete remission. In two patients, complete remission was preceded by striking leukocytosis that then resolved despite continued drug treatment. Serial studies of cellular morphologic features, cell-surface immunophenotypic analysis, and fluorescence in situ hybridization with a chromosome 17 probe revealed that clinical response was associated with maturation of the leukemic clone. All patients who responded to treatment who were tested by Northern blot analysis had expression of aberrant RAR-alpha. As patients entered complete remission, the expression of the abnormal RAR-alpha message decreased markedly; however, it was still detectable in several patients after complete morphologic and cytogenetic remission had been achieved. CONCLUSIONS Tretinoin is a safe and highly effective agent for inducing complete remission in patients with acute promyelocytic leukemia. Clinical response to this agent is associated with leukemic-cell differentiation and is linked to the expression of an aberrant RAR-alpha nuclear receptor. Molecular detection of the aberrant receptor may serve as a useful marker for residual leukemia in patients with this disease.

MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31-mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.

Retinoids in cancer therapy and chemoprevention: promise meets resistance

Retinoids (natural and synthetic derivatives of vitamin A) signal potent differentiation and growth-suppressive effects in diverse normal, premalignant, and malignant cells. A strong rationale exists for the use of retinoids in cancer treatment and chemoprevention based on preclinical, epidemiological, and early clinical findings. Despite the success of all-trans-retinoic acid (RA)-based differentiation therapy in acute promyelocytic leukemia (APL), the broad promise of retinoids in the clinic has not yet been realized. In addition to the expected limited activity of any single therapeutic agent, translation of retinoid activities from the laboratory to the clinic has met with intrinsic or acquired retinoid resistance. Evidence suggests that solid tumors develop intrinsic resistance to retinoids during carcinogenesis. In contrast, relapse of APL is often associated with acquired resistance to retinoid maturation induction. This review discusses what is known about retinoid resistance mechanisms in cancer therapy and chemoprevention. Strategies to overcome this resistance will be discussed, including combination therapy with other differentiation-inducing, cytotoxic or chromatin-remodeling agents, as well as the use of receptor-selective and nonclassical retinoids. Opportunities exist in the post-genomic era to bypass resistance to classical retinoids by identifying target genes and associated pathways that directly mediate the antineoplastic effects of retinoids. In this regard, the retinoids are useful pharmacological tools to reveal important pathways targeted in cancer therapy and chemoprevention.

All-trans Retinoic Acid for Acute Promyelocytic Leukemia: Results of the New York Study

Acute promyelocytic leukemia is a distinct clinical and pathologic subtype of acute myeloid leukemia that is characterized by reciprocal translocations between the long arms of chromosomes 15 and 17 [1, 2]. The breakpoint on chromosome 17 disrupts a gene that encodes a nuclear receptor for retinoic acid (RAR-) [3], and its translocation to chromosome 15 results in a fusion with a newly described gene called PML, which may act as a transcription factor [4-7]. Like conventional cytogenetic analysis, detection of PML/RAR- fusion mRNA is now used for the molecular diagnosis of this disease [8, 9]. Severe coagulopathy and a high incidence of early fatal hemorrhage characterize acute promyelocytic leukemia [10, 11]. The risk for hemorrhage is exacerbated further by cytotoxic chemotherapy, probably because of rapid cell lysis and release of intracellular procoagulants [12]. Despite the high initial mortality rate, patients with acute promyelocytic leukemia who achieve complete remission appear to have better long-term survival compared with patients who have other types of acute myeloid leukemia [13-19]. Recent studies in China, France, Japan, and the United States showed that all-trans retinoic acid induces complete remission in many patients with acute promyelocytic leukemia [20-24]. Because all-trans retinoic acid initially induces cytodifferentiation rather than immediate lysis of leukemic cells [21, 22], this agent theoretically should decrease the early mortality rate. However, if fewer leukemic cells were eliminated with all-trans retinoic acid than with cytotoxic drugs, such therapy might prove disadvantageous by increasing the proportion of patients who subsequently relapse. To evaluate the role of this drug in an integrated treatment strategy, we conducted a clinical study that used all-trans retinoic acid to induce remission and as maintenance therapy in patients with acute promyelocytic leukemia. Methods Patients Our study comprised consecutive patients treated with all-trans retinoic acid during a 2-year period at our institution from June 1990 through June 1992. (A report on the first 11 cases in this series was previously published [22].) We considered patients for this study if they fulfilled morphologic criteria of acute promyelocytic leukemia (M3 or M3-variant) according to the French-American-British classification [25]. Patients with newly diagnosed disease and those who had relapsed from cytotoxic chemotherapy were eligible. We required that women able to bear children have a negative pregnancy test and cautioned them to use effective contraceptive methods. All patients gave signed informed consent, and our centers institutional review board approved the study. Treatment To Induce Remission We administered all-trans retinoic acid in two divided oral doses of 45 mg/m2 per day (rounded up to the nearest 10 mg) after meals. Exceptions to this regimen occurred on days when patients underwent additional pharmacologic testing, in which case the drug was taken as a single morning dose; also, several patients received the drug in an oil-based suspension through a nasogastric tube because of endotracheal intubation. We withdrew patients who were shown by molecular testing to lack the PML/RAR- rearrangement and treated them with chemotherapy. Consolidation and Maintenance Treatment Plan Previous treatment status determined postremission therapy. Patients who were newly diagnosed continued to receive all-trans retinoic acid for approximately 30 days after they achieved complete clinical remission. We then discontinued the drug and administered three courses of consolidation chemotherapy that consisted of idarubicin and cytosine arabinoside. The first chemotherapy cycle was similar in dosage to conventional induction therapy in which idarubicin (12 mg/m2 per day given intravenously for 3 days) and cytosine arabinoside (200 mg/m2 per day given intravenously for 5 days) were administered concurrently. Subsequent cycles were repeated every 3 to 6 weeks, depending on patient tolerance, using idarubicin (at the same daily dosage for 2 days) and cytosine arabinoside (200 mg/m2 per day for 4 days). Five patients received a slightly different consolidation chemotherapy: Three received daunorubicin rather than idarubicin; one refused further treatment after only two of the three planned chemotherapy courses; and one pediatric patient received six cycles of chemotherapy at lower dosages. Two other newly diagnosed patients did not receive chemotherapy (because of advanced age and patient refusal); rather, they received only all-trans retinoic acid. The initial cohort of patients who relapsed from previous chemotherapy-induced remissions were given all-trans retinoic acid therapy unless they were eligible for bone marrow transplant. When we found that remissions maintained by all-trans retinoic acid were brief, a subsequent cohort of previously treated patients received consolidation therapy using a radionuclide-conjugated monoclonal antibody [26]. Management of Leukocytosis, the Retinoic Acid Syndrome, and Coagulopathy An early report indicated that patients in whom leukocytosis developed fared poorly [27], and management of this complication evolved during the course of these studies. Although most patients received no specific treatment for leukocytosis, we used leukapheresis for five patients who had rapid increases in their peripheral blood leukocyte counts; intravenous infusions of cytosine arabinoside (100 mg/m2 per day for several days) were also administered to five patients. After we recognized a distinctive respiratory distress syndrome as a specific complication of all-trans retinoic acid therapy [28], we gave patients intravenous dexamethasone (10 mg every 12 hours for 3 to 5 days) as prophylactic treatment at the earliest clinical sign of dyspnea. Contrary to a previously routine practice at this center and elsewhere, generally we did not use heparin to treat disseminated intravascular coagulation. Instead, patients with laboratory evidence of coagulopathy received frequent (up to twice daily) transfusions of platelets and fresh frozen plasma to maintain the platelet count at more than 50 109/L and the fibrinogen level at more than 1.0 g/L. We reserved heparin for patients with thrombosis or refractory coagulopathy. Follow-up during the Study Patients were evaluated each day as inpatients during the initial phase of the study and later as outpatients twice each week until complete remission was achieved. During remission induction, serial assessments were done for blood cell counts, serum biochemical values, and coagulation parameters. In early patients, bone marrow aspiration was done every 5 to 10 days, but this was reduced in later patients. Cytogenetic studies were done using Giemsa or quinacrine banding techniques. The RNA extracted from the mononuclear cell fraction of bone marrow aspirates was analyzed using a reverse transcription polymerase chain reaction assay for the PML/RAR- rearrangement [9]. We observed conventional response criteria [29] and assessed response and toxicity in all patients. Selection of Historical Controls We compared newly diagnosed patients who entered this study during a 2-year period with a group of historical control patients. The control group consisted of 80 consecutive patients with newly diagnosed acute promyelocytic leukemia who were treated in protocols at our center during the 15-year period that immediately preceded initiation of this study. Induction chemotherapy in the historical control group consisted of cytosine arabinoside combined with daunorubicin (n = 50), idarubicin (n = 18), or amsacrine (n = 12). The median age of this group was 34 years (range, 15 to 71 years). Other aspects of the control patients were described in two recent reports [13, 30]. Statistical Methods We estimated the probability of event-free and overall survival using the Kaplan-Meier method. Results Patient Characteristics We entered into our study 56 consecutive patients admitted to our center for acute promyelocytic leukemia. Thirty-four patients were newly diagnosed and 22 had relapsed from one or more chemotherapy-induced remissions. The clinical characteristics of these patients are summarized in Table 1. We identified the 15; 17 chromosomal translocation in 51 of 56 patients by cytogenetic study or by molecular analysis of the PML/RAR- rearrangement. Five of the 56 patients had a normal cytogenetic karyotype and no mRNA expression of PML/RAR-; their leukemic cells showed no evidence of maturation with all-trans retinoic acid treatment, and we withdrew them from the study when we obtained the molecular result. Five patients entered the study after treatment for another cancer: three for breast cancer, one for Hodgkin disease (each of whom had previously received cytotoxic chemotherapy), and one for prostate cancer. The median initial leukocyte count in the peripheral blood was 1.9 109/L (range, 0.4 to 147.7 109/L). Four newly diagnosed patients and three patients who had relapsed had M3-variant morphology. Laboratory findings consistent with disseminated intravascular coagulation were detected in 38 of the 56 patients at the time of entry. Table 1. Clinical Characteristics of Patients with Acute Promyelocytic Leukemia Treated with All-trans Retinoic Acid for Remission Induction* Response As shown in Table 2, 44 of the 51 patients (86%; 95% CI, 76% to 96%) with cytogenetic or molecular evidence of acute promyelocytic leukemia achieved complete remission with all-trans retinoic acid treatment. In patients with PML/RAR- rearrangements, the response in newly diagnosed patients (26 of 30 cases) was similar to that of previously treated patients (18 of 21 cases). We removed 1 previously treated patient early in the study because of asymptomatic leukocytosis, and she achieved remission with full-dose chemotherapy; however, we classified her as a nonresponder for this study. We interrup

Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A

The PLZF gene was identified by its fusion with the RARα locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARα-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARα-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.

Retinoic Acid Promotes Ubiquitination and Proteolysis of Cyclin D1 during Induced Tumor Cell Differentiation

Mechanisms by which differentiation programs engage the cell cycle are poorly understood. This study demonstrates that retinoids promote ubiquitination and degradation of cyclin D1 during retinoid-induced differentiation of human embryonal carcinoma cells. In response to all-trans-retinoic acid (RA) treatment, the human embryonal carcinoma cell line NT2/D1 exhibits a progressive decline in cyclin D1 expression beginning when the cells are committed to differentiate, but before onset of terminal neuronal differentiation. The decrease in cyclin D1 protein is tightly associated with the accumulation of hypophosphorylated forms of the retinoblastoma protein and G1 arrest. In contrast, retinoic acid receptor γ-deficient NT2/D1-R1 cells do not growth-arrest or accumulate in G1 and have persistent cyclin D1 overexpression despite RA treatment. Notably, stable transfection of retinoic acid receptor γ restores RA-mediated growth suppression and differentiation to NT2/D1-R1 cells and restores the decline of cyclin D1. The proteasome inhibitor LLnL blocks this RA-mediated decline in cyclin D1. RA treatment markedly accelerates ubiquitination of wild-type cyclin D1, but not a cyclin D1 (T286A) mutant. Transient expression of cyclin D1 (T286A) in NT2/D1 cells blocks RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and represses induction of immunophenotypic neuronal markers. Taken together, these findings strongly implicate RA-mediated degradation of cyclin D1 as a means of coupling induced differentiation and cell cycle control of human embryonal carcinoma cells.

Uncovering Growth-Suppressive MicroRNAs in Lung Cancer

Purpose: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer. Experimental Design: miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal-malignant human lung tissue bank. Results: miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1 and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided a mechanism for observed growth suppression. Conclusions: miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.

Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

The synthetic triterpenoids CDDO-methyl ester and CDDO-ethyl amide prevent lung cancer induced by vinyl carbamate in A/J mice.

We report the first use of new synthetic triterpenoids to prevent lung cancer in experimental animals. Female A/J mice were treated with the mutagenic carcinogen vinyl carbamate, which induces adenocarcinoma of the lung in all animals within 16 weeks. If mice were fed either the methyl ester or the ethyl amide derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-ME and CDDO-EA, respectively), beginning 1 week after dosing with carcinogen, the number, size, and severity of lung carcinomas were markedly reduced. The mechanisms of action of CDDO-ME and CDDO-EA that are germane to these in vivo findings are the following results shown here in cell culture: (a) suppression of the ability of IFN-gamma to induce de novo formation of nitric oxide synthase in a macrophage-like cell line RAW264.7, (b) induction of heme oxygenase-1 in these RAW cells, and (c) suppression of phosphorylation of the transcription factor signal transducers and activators of transcription 3 as well as induction of apoptosis in human lung cancer cell lines.

UBE1L is a retinoid target that triggers PML/RARα degradation and apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)/RA receptor α (RARα). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML/RARα, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of retinoid response in APL is the proteasome-dependent PML/RARα degradation. UBE1L transfection triggered PML/RARα degradation, but transfection of a truncated UBE1L or E1 did not cause this degradation. A tight link was shown between UBE1L induction and PML/RARα degradation. Notably, retroviral expression of UBE1L rapidly induced apoptosis in NB4 APL cells, but not in cells lacking PML/RARα expression. UBE1L has been implicated directly in retinoid effects in APL and may be targeted for repression by PML/RARα. UBE1L is proposed as a direct pharmacological target that overcomes oncogenic effects of PML/RARα by triggering its degradation and signaling apoptosis in APL cells.