FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL.

Targeted delivery of antisense oligodeoxynucleotide by transferrin conjugated pH-sensitive lipopolyplex nanoparticles: a novel oligonucleotide-based therapeutic strategy in acute myeloid leukemia.

Therapeutic use of oligodeoxynucleotides (ODNs) that hybridize to and downregulate target mRNAs encoding proteins that contribute to malignant transformation has a sound rationale, but has had an overall limited clinical success in cancer due to insufficient intracellular delivery. Here we report a development of formulations capable of promoting targeted delivery and enhanced pharmacologic activity of ODNs in acute myeloid leukemia (AML) cell lines and patient primary cells. In this study, transferrin (Tf) conjugated pH-sensitive lipopolyplex nanoparticles (LPs) were prepared to deliver GTI-2040, an antisense ODN against the R2 subunit of ribonucleotide reductase that has been shown to contribute to chemoresistance in AML. LPs had an average particle size around 110 nm and a moderately positive zeta potential at approximately 10 mV. The ODN encapsulation efficiency of LPs was >90%. These nanoparticles could release ODNs at acidic endosomal pH and facilitate the cytoplasmic delivery of ODNs after endocytosis. In addition, Tf-mediated targeted delivery of GTI-2040 was achieved. R2 downregulation at both mRNA and protein levels was improved by 8-fold in Kasumi-1 cells and 2- to 20-fold in AML patient primary cells treated with GTI-2040-Tf-LPs, compared to free GTI-2040 treatment. Moreover, Tf-LPs were more effective than nontargeted LPs, with 10 to 100% improvement at various concentrations in Kasumi-1 cells and an average of 45% improvement at 3 microM concentration in AML patient primary cells. Treatment with 1 microM GTI-2040-Tf-LPs sensitized AML cells to the chemotherapy agent cytarabine, by decreasing its IC(50) value from 47.69 nM to 9.05 nM. This study suggests that the combination of pH sensitive LP formulation and Tf mediated targeting is a promising strategy for antisense ODN delivery in leukemia therapy.

Phase I study of GTI-2040, an antisense to ribonucleotide reductase, in combination with high-dose cytarabine in patients with acute myeloid leukemia.

PURPOSE: Inhibition of ribonucleotide reductase reduces the availability of the endogenous pool of deoxycytidine and may increase cytarabine (AraC) cytotoxicity. We performed a phase I dose escalation trial of AraC combined with GTI-2040, a 20-mer antisense oligonucleotide shown in preclinical studies to decrease levels of the R2 subunit of ribonucleotide reductase, to determine the maximum tolerated dose in adults with relapsed/refractory acute myeloid leukemia. EXPERIMENTAL DESIGN: Twenty-three adults (ages 18-59 years) were enrolled in this dose escalation phase I trial, receiving high-dose AraC twice daily combined with infusional GTI-2040. An ELISA-based assay measured plasma and intracellular concentrations of GTI-2040. R2 protein changes were evaluated by immunoblotting in pretreatment and post-treatment bone marrow samples. RESULTS: The maximum tolerated dose was 5 mg/kg/d GTI-2040 (days 1-6) and 3 g/m2/dose AraC every 12 hours for 8 doses. Neurotoxicity was dose limiting. Eight patients (35%) achieved complete remission. Mean bone marrow intracellular concentration of GTI-2040 were higher at 120 hours than at 24 hours from the start of GTI-2040 (P = 0.002), suggesting intracellular drug accumulation over time. Reductions in bone marrow levels of R2 protein (>50%) were observed at 24 and 120 hours. Higher baseline R2 protein expression (P = 0.03) and reductions after 24 hours of GTI-2040 (P = 0.04) were associated with complete remission. CONCLUSIONS: GTI-2040 and high-dose AraC were coadministered safely with successful reduction of the intended R2 target and encouraging clinical results. The clinical efficacy of this combination will be tested in an upcoming phase II study.

Interplay of RUNX1/MTG8 and DNA methyltransferase 1 in acute myeloid leukemia.

The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML.

Phase I study of GTI-2040, a ribonucleotide reductase antisense, with high dose cytarabine in patients with relapsed/refractory acute myeloid leukemia.

We hypothesized that GTI-2040, a 20-mer oligonucleotide complementary to the R2 subunit mRNA of ribonucleotide reductase, combined with high dose cytarabine (HiDAC) would result in enhanced cytotoxicity by favoring Ara-CTP DNA incorporation. In a phase I dose escalation trial, adults (≥ 60 years) with refractory or relapsed acute myeloid leukemia (AML) received daily HiDAC plus infusional GTI-2040. Using a novel assay, evidence of intracellular drug accumulation and target R2 down-regulation was observed. GTI-2040/HiDAC can be administered safely. However, with no complete remissions observed, alternative doses and schedules may need to be investigated to achieve clinical activity in older patients with AML.

Linking tRNA localization with activation of nutritional stress responses.

Cells respond to nutrient deprivation a variety of ways. In addition to global downregulation of cap-dependent protein synthesis mediated by the GCN2 and mTORC1 signaling pathways, a catabolic process autophagy is upregulated to provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show reduced mTORC1 activity and upregulated autophagy. This suggests that subcellular localization of tRNAs may regulate an intracellular response to starvation independently of the cellular nutritional status.

Bortezomib induces DNA hypomethylation and silenced gene transcription by interfering with Sp1/NF-kappaB-dependent DNA methyltransferase activity in acute myeloid leukemia.

Bortezomib reversibly inhibits 26S proteasomal degradation, interferes with NF-kappaB, and exhibits antitumor activity in human malignancies. Zinc finger protein Sp1 transactivates DNMT1 gene in mice and is functionally regulated through protein abundance, posttranslational modifications (ie, ubiquitination), or interaction with other transcription factors (ie, NF-kappaB). We hypothesize that inhibition of proteasomal degradation and Sp1/NF-kappaB-mediated transactivation may impair aberrant DNA methyltransferase activity. We show here that, in addition to inducing accumulation of polyubiquitinated proteins and abolishment of NF-kappaB activities, bortezomib decreases Sp1 protein levels, disrupts the physical interaction of Sp1/NF-kappaB, and prevents binding of the Sp1/NF-kappaB complex to the DNMT1 gene promoter. Abrogation of Sp1/NF-kappaB complex by bortezomib causes transcriptional repression of DNMT1 gene and down-regulation of DNMT1 protein, which in turn induces global DNA hypomethylation in vitro and in vivo and re-expression of epigenetically silenced genes in human cancer cells. The involvement of Sp1/NF-kappaB in DNMT1 regulation is further demonstrated by the observation that Sp1 knockdown using mithramycin A or shRNA decreases DNMT1 protein levels, which instead are increased by Sp1 or NF-kappaB overexpression. Our results unveil the Sp1/NF-kappaB pathway as a modulator of DNA methyltransferase activity in human cancer and identify bortezomib as a novel epigenetic-targeting drug.

Targeting AML1/ETO-histone deacetylase repressor complex: a novel mechanism for valproic acid-mediated gene expression and cellular differentiation in AML1/ETO-positive acute myeloid leukemia cells.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting class I histone deacetylase (HDAC)-containing repressor complex to the promoter of AML1 target genes. Valproic acid (VPA), a commonly used antiseizure and mood stabilizer drug, has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition. VPA causes selective proteasomal degradation of HDAC2 but not other class I HDACs (i.e., HDAC 1, 3, and 8). Therefore, we raised the question of whether this drug can effectively target the leukemogenic activity of the AML1/ETO fusion protein that also recruits HDAC1, a key regulator of normal and aberrant histone acetylation. We report here that VPA treatment disrupts the AML1/ETO-HDAC1 physical interaction, stimulates the global dissociation of AML1/ETO-HDAC1 complex from the promoter of AML1/ETO target genes, and induces relocation of both AML1/ETO and HDAC1 protein from nuclear to perinuclear region. Furthermore, we show that mechanistically these effects associate with a significant inhibition of HDAC activity, histone H3 and H4 hyperacetylation, and recruitment of RNA polymerase II, leading to transcriptional reactivation of target genes (i.e., IL-3) otherwise silenced by AML1/ETO fusion protein. Ultimately, these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis. Taken together, these data support the notion that VPA might effectively target AML1/ETO-driven leukemogenesis through disruption of aberrant HDAC1 function and that VPA should be integrated in novel therapeutic approaches for AML1/ETO-positive AML.

Phase I study of decitabine alone or in combination with valproic acid in acute myeloid leukemia.

PURPOSE: To determine an optimal biologic dose (OBD) of decitabine as a single agent and then the maximum-tolerated dose (MTD) of valproic acid (VA) combined with decitabine in acute myeloid leukemia (AML). PATIENTS AND METHODS: Twenty-five patients (median age, 70 years) were enrolled; 12 were untreated and 13 had relapsed AML. To determine an OBD (based on a gene re-expression end point), 14 patients received decitabine alone for 10 days. To determine the MTD, 11 patients received decitabine (at OBD, days 1 through 10) plus dose-escalating VA (days 5 through 21). RESULTS: The OBD of decitabine was 20 mg/m(2)/d intravenously, with limited nonhematologic toxicity. In patients treated with decitabine plus VA, dose-limiting encephalopathy occurred in two of two patients at VA 25 mg/kg/d and one of six patients at VA 20 mg/kg/d. Drug-induced re-expression of estrogen receptor (ER) was associated with clinical response (P < or = .05). ER promoter demethylation, global DNA hypomethylation, depletion of DNA methyltransferase enzyme, and histone hyperacetylation were also observed. In an intent-to-treat analysis, the response rate was 44% (11 of 25). Of 21 assessable patients, 11 (52%) responded: four with morphologic and cytogenetic complete remission (CR; each had complex karyotype), four with incomplete CR, and three with partial remission. In untreated AML, four of nine assessable patients achieved CR. Clinical responses appeared similar for decitabine alone or with VA. CONCLUSION: Low-dose decitabine was safe and showed encouraging clinical and biologic activity in AML, but the addition of VA led to encephalopathy at relatively low doses. On the basis of these results, additional studies of decitabine (20 mg/m(2)/d for 10 days) alone or with an alternative deacetylating agent are warranted.