Combination of Epigenetic Agents Synergistically Reverse the Malignant Phenotype in Models of T-Cell Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2727-2727 ◽  
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle C Bongero ◽  
Christine M McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Combination Group ◽  
T Cell Lymphomas

Abstract Abstract 2727 CHOP and CHOP-like chemotherapy remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL) and peripheral T-cell lymphomas (PTCL) given their marked single agent activity in these diseases. The interaction between the HDACIs (depsipeptide (R) and belinostat (B)) and a DNMT inhibitor (decitabine (D)) was investigated in vitro, in vivo and at the molecular level in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (P12, PF-382). For all cytotoxicity assays, a luminescence based cell viability assay was used (CellTiter-Glo™) followed by acquisition on a Biotek Synergy HT. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition and analyzed using FlowJo. The IC50s for B, R, vorinostat (V), panobinostat (P), D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours in all the cell lines. For the combination experiment we selected the most active DNMTI, decitabine. In the cytotoxicity assays, the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines studied. The RRRs for all the combinations were between 0.0007 and 0.9. When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 1 displays the range of apoptosis induction for B, R ± D and the RRR value for the most significant data.Table 1:BDB + DRRR(% Apoptotic + Dead Cells)H9100 nM (22.9%)500 nM (17.9%)51.5%0.7HH100 nM (42.9%)1 uM (46.9%)61.3%0.8P 12150 nM (16%)1 uM (42.7%)80.1%0.4PF 382100 nM (8.3%)1 uM (27.9%)40.1%0.8RDR + DH92 nM (22.2%)500 nM (17.9%)63.6%0.5HH2 nM (80%)1 uM (46.9%)89.7%0.6P 122 nM (9.9%)10 uM (58.7%)98%0.03PF 3822 nM (54.5%)500 nM (17.9%)88.7%0.2 An in vivo xenograft study in 6–8 weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells was performed. Mice were separated into different cohorts and treated i.p. for 3 cycles with D or B or their combination according to the following schedules: D at 1.5 mg/kg on days 1, 3, 5; B at 40 mg/kg/day for 10 days (I cycle); D at 1.5mg/kg on days 15,17,19,21; B at 65 mg/kg/day for 10 days (II cycle); D at 1.5 mg/kg on days 29,31,33,35,37,39,41,43; B at 100mg/kg for 19 days (III cycle). Statistically significantly tumor growth inhibition was observed in the combination cohort compared to all the other cohorts (analysis on day 42, 45). We analyzed the molecular basis for this synergistic effect by evaluating gene expression patterns using the Illumina Human HT-12 v4 Expression BeadChip microarrays. These analyses revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. As shown in Figure 1, a set of genes (A) is down-regulated by both drugs. Other genes (B) are up-regulated by D and the effect is maintained in the combination. Other genes (C+E) are slightly up-regulated by R, though not significantly modified by D, and more strongly up-regulated in the combination group. Similarly, genes to some extent up-regulated by D but not by R (D+F) appeared to be more significantly affected by the combination. As shown in Figure 2, the effects of the two drugs are largely different (only 39 genes modified in common by all the treatment groups). Most of the effects induced by the single agent treatment are maintained in the combination group (174 genes out of 191 for romidepsin and 211 genes out of 221 for decitabine). Interestingly, an additional 944 genes appeared to be modulated uniquely by the combination treatment strongly supporting the hypothesis of synergism also at the molecular level. Collectively, the data suggest that the combination of a DNMTI and HDACIs is synergistic in in vitro and in vivo model of T-cell lymphoma and is able to synergistically reverse the malignant signature at the molecular level. These data may constitute the basis for future phase I-II clinical trials. Disclosures: O'Connor: celgene: Consultancy, Research Funding; merck: Research Funding; Novartis: Research Funding; spectrum: Research Funding.

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13569-e13569
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle Bongero ◽  
Christine McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

T-Cell Lymphoma Patient-Derived Xenografts and Newly Developed Cell Lines Recapitulate Aspects of Disease Biology and Represent Novel Tools for Preclinical Drug Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3015-3015 ◽  
Author(s):  
Amanda L. Christie ◽  
Samuel Y. Ng ◽  
Raphael Koch ◽  
Alexandra N. Christodoulou ◽  
Tiffany DeSouza ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Renal Capsule ◽  
Peripheral T Cell Lymphoma ◽  
In Vivo Models ◽  
T Cell Lymphomas

Abstract Lymphomas represent nearly 70 distinct diseases with unique clinical presentations, therapeutic responses and underlying biology. There is a pressing shortage of publically available cell line and in vivo models of nearly all of these diseases; T-cell lymphoma models are particularly under-represented compared to B-cell lymphomas, which has severely hampered efforts to understand and target their biology. The majority ofin vivo models of T-cell lymphomas are genetically-engineered mouse models, which often don't faithfully recapitulate human disease. To address this issue, we have established a repository of patient-derived xenografts (PDX) of lymphomas by engrafting human tumors into immunodeficient NOD/Scid/IL2rgnull mice with or without an MHC Class 1 deficiency (to prevent graft versus host disease). Blood and bone marrow specimens involved with tumor were injected by tail vein injection. Lymph node and extranodal biopsy specimens were implanted under the renal capsule as a 1x1x2mm tumor seed, which maintains the in situ microarchitecture. A description of T-cell lymphoma PDXs is included in the Table. PDXs have been extensively characterized by immunohistochemistry (IHC), flow cytometry, transcriptome sequencing and targeted DNA sequencing. These studies have demonstrated retention of key architectural, cellular, and molecular features of the primary tumors. Flow cytometric analysis of patient tumors and their respective xenografts revealed highly concordant patterns of surface marker expression. IHC of murine tissues confirmed retention of tumor immunophenotypes, architecture, and even tissue tropism in the PDXs. For example, blood from a patient with Sézary Syndrome manifested in the skin of recipient mice when injected into the lateral tail vein. A breast implant-associated ALK-negative anaplastic large cell lymphoma (ALCL) implanted under the renal capsule metastasized to the liver and spleen while uniformly retaining CD30 positivity. A peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) specimen implanted under the renal capsule engrafted in the spleen, with the notable admixture of nonmalignant T cells and scattered EBV-positive B cells in first passage. T-cell receptor gene rearrangement PCR performed on this PTCL-NOS demonstrated an identical rearrangement pattern in the primary tumor and the PDX. An angioimmunoblastic T-cell lymphoma (AITL) specimen engrafted in spleen, lymph node and bone marrow within 6 weeks and serially transplanted through three generations in an orthotopic manner while maintaining a CD3+CD4+PD1+CD30partial immunophenotype. The genetic characterization of the PDX models using a targeted DNA sequencing approach showed a mutational profile that clearly matched primary T-cell lymphoma samples and significantly expands the current repertoire of available pre-clinical models. For example, a PDX model of AITL showed mutations of TET2 and ARID1B; a model of an ALK-negative ALCL harbored mutations of STAT3 and STAT5. This massively extends the spectrum of clinically representative model systems that can be used to explore novel therapeutic strategies for T-cell lymphomas. Several early-passage PDXs have been used to generate T-cell lymphoma cells lines, including three cell lines from AITL PDX models. One of these AITL cell lines has proliferated through 30 passages and was validated by immunophenotype and molecular confirmation of bi-allelic TET2 mutations with loss of 6q, 7q, and 10q confirmed using Sanger and TruSeq Custom Amplicon Sequencings. To our knowledge, there have been no reports of an AITL cell line in the literature. Additional peripheral T-cell lymphoma cell lines are currently under development. These lymphomas, along with a spectrum of PDXs of other hematologic malignancies, are available to collaborators through the online portal PRoXe (Public Repository of Xenografts) at www.proxe.org. These models represent a unique opportunity to interrogate biology and perform preclinical studies with in vivo models. Table 1 Table 1. Disclosures Jacobson: Kite: Membership on an entity's Board of Directors or advisory committees. Armand:Pfizer: Research Funding; Sequenta Inc: Research Funding; Merck: Consultancy, Research Funding; Roche: Research Funding; Infinity Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Shipp:Bristol-Myers Squibb: Consultancy, Research Funding; Cell Signaling: Honoraria; Merck, Gilead, Takeda: Other: Scientific Advisory Board; Bayer: Research Funding. Fisher:Pharmacyclics: Consultancy. Weinstock:Novartis: Consultancy, Research Funding.

scholarly journals Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 720-720
Author(s):  
Akihiro Kitadate ◽  
Sho Ikeda ◽  
Fumito Abe ◽  
Naoto Takahashi ◽  
Norio Shimizu ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Cell Lymphoma ◽  
Selective Inhibitor ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Class I ◽  
T Cell Lymphomas

Abstract Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice. Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6). Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat. Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. Disclosures Kitadate: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa: TaNeDS (Daiichi Sankyo): Research Funding.

scholarly journals Venetoclax Synergizes with Radiation Therapy for Treatment of B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 467-467
Author(s):  
Shyril O'Steen ◽  
Amelia Waltman ◽  
Garrett Booth ◽  
Aimee L Kenoyer ◽  
Margaret Nartea ◽  
...  
Keyword(s):  
B Cell ◽  
Survival Time ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Combination Group ◽  
Mean Survival Time

Abstract Introduction: An estimated 19,970 Americans died of non-Hodgkin lymphoma (NHL) in 2015, with diffuse large B-cell lymphoma (DLBCL) accounting for roughly 30% of newly diagnosed NHL. Our study focuses on three NHL subtypes: germinal center (GCB)-DLBCL, the most common DLBCL subtype; activated (ABC)-DLBCL, a particularly aggressive and high-risk subtype; and mantle cell lymphoma (MCL), considered incurable. Constitutive B-cell receptor signaling is implicated in the pathogenesis of ABC-DLBCL and MCL and may couple with aberrant apoptotic BCL-2 pathway proteins. The BCL-2 inhibitor venetoclax is a promising targeted agent that promotes apoptosis in a variety of NHL subtypes, but is almost never curative as a single agent. Radiotherapy promotes apoptosis by creating DNA strand breaks, and we hypothesized that the combination of radiotherapy and venetoclax would act synergistically in NHL to increase the probability of cures. Methods: We tested in vitro killing efficacy of sublethal 137Cesium irradiation combined with venetoclax in 15 cell lines, representing a diversity of NHL subtypes. Cells were treated with 137Cesium and venetoclax in 8 x 8 dose combination matrices, incubated 72-120 hrs, then assayed for viability with Celltiter-Glo (Promega). The degree of treatment antagonism, additivity, or synergism was determined using isobolographic analyses. For in vivo studies, we tested combinations of venetoclax with either 137Cesium total body irradiation (TBI), or CD20 pre-targeted radioimmunotherapy (PRIT), in threetumor models chosen for divergent single agent sensitivities. Tumor xenografts of Rec-1 (MCL), U2932 (ABC-DLBCL), and SU-DHL-6 (GCB-DLBCL) were produced by subcutaneous flank injection of 10 x 106 cells in male and female NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice. When tumor volumes were 50 mm3, mice (n = 8-12/group) were treated with either venetoclax (100-200mg/kg daily for 10-30 days), diluent control, TBI (single dose, 6-10 Gy 137Cesium), or a combination of venetoclax and TBI. In PRIT studies, mice were coinjected with 300µg unlabeled streptavidin-conjugated anti-CD20 antibody (murine IgG2a) and 400µg HB8181 (IgG2a isotype control to block non-specific binding) in place of TBI. Twenty-one hours later, 5.8 nmol biotin-galactose "clearing agent" was administered, followed in 3 hours by 1.2 nmol DOTA-biotin labeled with 400, 800, or 1200 µCi of 90Y (14.8, 29.6, or 44.4 MBq, respectively). Results: In vitro, 10 of 15 lymphoma cell lines responded synergistically to combined radiotherapy and venetoclax, including GCB-DLBCL, ABC-DLBCL and MCL lines (p < .04 in 10 cell lines). In vivo, each of 3 lymphoma models responded synergistically to combination therapy. In mice bearing Rec-1 xenografts, venetoclax alone did not affect mean survival time (p = .32), 8 Gy TBI lengthened survival by 44% compared to controls (p < .0001), but TBI combined with venetoclax tripled survival time compared to controls (p < .0001, combination group > TBI alone). The SU-DHL-6 model produced similar results. In the U2932 model, tumors disappeared during venetoclax monotherapy, but recurred in all mice, such that mean survival time doubled compared to controls (p = .0001). Six Gy TBI had no effect (p = .73), but combining TBI with venetoclax tripled survival time compared to controls (p = .0003, combination group > venetoclax alone). Using PRIT in place of TBI produced yet greater efficacy. In Rec-1 bearing mice, venetoclax had no effect alone (p = .12), 800µCi PRIT lengthened survival time 111% beyond controls (p = .0001), while the combination extended survival 483% beyond controls and cured 40% (p = .001, combination group > PRIT alone). In the U2932 xenograft model, venetoclax alone doubled survival time compared to controls (p < .0001) and 800µCi PRIT alone doubled survival and cured 30% (Fig. 1, p < .0001). Combination treatments cured 100% (Fig. 1). Conclusion: In vitro and in vivo results support our hypothesis that radiotherapy combines effectively with venetoclax to treat NHL. Despite differences in single agent sensitivity, xenograft models of GCB-DLBCL, ABC-DLBCL and MCL all responded synergistically to combinations of either TBI or PRIT with venetoclax. PRIT combinations with venetoclax produced cures (Fig. 1) without detectable toxicity, and merit clinical preference. Ongoing studies examine predictive biomarkers and optimal treatment protocols for therapeutic efficacy. Disclosures Gopal: Paid Consultancy- Gilead, Janssen, Seattle Genetics, Spectrum, Research funding- Gilead, Janssen, Pfizer, BMS, Merck, Teva, Takeda, Spectrum, Seattle Genetics: Consultancy, Honoraria, Research Funding.

scholarly journals The Novel Tumor Suppressor SAMHD1 Is Differentially Expressed and Partly Regulated By MYC in Peripheral T-Cell Lymphomas (PTCL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4130-4130
Author(s):  
Pedro Farrajota Neves Da Silva ◽  
Nikolaos Tsesmetzis ◽  
Ioanna Xagoraris ◽  
Agata Magdalena Wasik ◽  
Georgia Kokaraki ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Differentially Expressed ◽  
Lymphoma Cells ◽  
T Cell Lymphomas ◽  
Samhd1 Expression

Abstract Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates required for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. More recently, SAMHD1 mutations have been reported in T-prolymphocytic leukemia (T-PLL). Based on these findings and the fact that SAMHD1 limits the dNTP pool in the cell, it may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, with cytarabine in acute myeloid leukemia being an example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of peripheral T-cell lymphomas (PTCL) are not yet known. Methods: The patient cohort included 64 PTCLs of various histologic types which were diagnosed and treated at Karolinska University Hospital (Sweden). A control group of 4 reactive lymph nodes and 2 reactive tonsils was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on a PTCL tissue microarray (TMA) with duplicate tumor cores from each case or full tissue sections using dual immunostaining (SAMHD1 / CD68) and a monoclonal antibody against SAMHD1 (Bethyl Laboratories, San Antonio, TX). At least 500 lymphoma cells were counted to calculate the percentage of SAMHD1-positive tumor cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. Two T-cell lymphomas cell lines (Mac1, Mac2A) were used as an in vitro system. As our preliminary findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, we hypothesized that MYC might regulate SAMHD1 expression. Therefore, the T-cell lymphoma cell lines were treated with the selective BET / MYC inhibitor JQ-1 or transiently transfected with a MYC-overexpressing plasmid or MYC gene-specific siRNA constructs, respectively. Western blot analysis was used to assess the protein levels. Results: SAMHD1 protein was expressed in reactive T-cells and histiocytes (CD68+) in all reactive lymphoid tissues (lymph nodes and tonsils) with strong staining intensity. SAMHD was differentially expressed among PTCL subtypes generally with weaker staining intensity as compared to normal T-cells and histiocytes, thus being positive in all (100%) angioimmunoblastic T-cell lymphomas (AILT), 67% PTCL-NOS, 45% ALK+ ALCL, 20% of ALK+ ALCL, and none (0%) of T-lymphoblastic lymphomas (p=0.0017, chi-square test). Among the SAMHD1- positive cases, the percentage of positive lymphoma cells ranged from 0 to 100% and its highest median was observed in AILT. SAMHD1 expression inversely correlated with CD30 expression (% CD30+ positive lymphoma cells) (p=0.0025, Mann-Whitney test). No significant associations between SAMHD1 levels and other clinicopathologic parameters or clinical outcome (EFS or OS) were found, however, the number of patients analyzed in each histologic subtype was limited. Inhibition of MYC activity by JQ-1 or MYC gene silencing with specific siRNA resulted in a substantial increase in the SAMHD1 protein level in T-cell lymphoma cell lines. Inversely, transient transfection of the cell lines with a MYC overexpressing plasmid resulted in decreased levels of SAMHD1. Taken together, the in vitro data suggest a possible MYC-associated regulation (repression) of SAMHD1 gene expression in T-cell lymphoma. Conclusions: SAMHD1 is shown for the first time to be differentially expressed among PTCL types and its regulation may involve MYC. Preliminary survival analysis shows no significant associations of SAMHD1 expression with EFS and OS in this cohort of PTCL, however, analysis of a larger PTCL study group is underway to draw definite conclusions. Disclosures Österborg: Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding.

scholarly journals GSK458 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in T Cell Lymphomas As a Single Agent and in Combination Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5378-5378 ◽  
Author(s):  
Juan Gu ◽  
Lianjuan Yang ◽  
Dennis C Gaughan ◽  
Ling He ◽  
Weina Shen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
T Cell Lymphoma ◽  
Synergistic Activity ◽  
T Cell Lymphomas

Abstract Introduction: T-cell lymphoma (TCL) accounts approximately 15% of all Non-Hodgkin's lymphoma cases. TCL are often divided into either systemic or cutaneous TCL (CTCL). The management of TCL systemic or cutaneous is challenging because the emergence of chemotherapy resistance that lead to early death (systemic T-cell lymphomas) or chronic debilitating clinical course (CTCL). To improve the clinical outcomes and reduce treatment-related toxicity, research need to be done focusing on understanding and targeting the molecular mechanism driving TCL subtypes. Recently, studies showed that the PI3K AKT/mTOR pathway was activated in TCL. GSK458 is a potent oral dual inhibitor of pan PI3K (α, β, γ and δ) and mTOR (mTOR1 and mTOR2). Preclinical studies in B-cell lymphomas showed GSK458 had broad antitumor activity in vitro and in vivo. In 2016, Phase I clinical trial of GSK458 was competed at the maximal dosage of 2.5mg twice daily. However, the effects of GSK458 on T-cell malignancies remain utterly unknown. Here, we evaluated the activity of GSK458 in preclinical T cell lymphoma models. Methods: We used a panel of T-cell lymphoma cell lines representing PTCL (J45), T-cell lymphoblastic lymphoma (SupT-1), and Mycosis Fungoides (MF)(MJ, HH and H9). TCL cell lines were exposed to escalating doses of GSK458 (1nM-100µM) without or with chemotherapeutic agents (doxorubicin, cisplatin, carboplatin, and dexamethasone); Bcl-2 inhibitor (Venetoclax); proteasome inhibitors ( bortezomib, carfilzomib, Ixazomib); or HDAC inhibitors (SAHA) for 48 and 72 hrs. Differences in cell viability, ATP levels, low mitochondria potential, glucose update, apoptosis and cell cycle distribution were evaluated utilizing PrestoBlue, Cell-Titer Glo assays, DiOC6, 2-NDG, Annexin V and propidium iodide staining followed by flow cytometric analysis, respectively. IC50 was calculated by GraphPad. PI3K and mTOR downstream pathway phosphorylation status, such as p-AKT Ser473, p-AKT Thr308, p-mTOR and p-GSK3β were detected by internally staining of FITC conjugated-antibodies followed by flow cytometry. Apoptosis proteins (MCL-1, PARP, p53, XIAP etc.) were detected by western blot. The additive/synergistic activity of GSK458 was detected by presto blue assay and Coefficient of synergy was calculated using CalcuSyn. Results:In vitro exposure of TCL cell lines to GSK458 demonstrated a dose- and time-dependent cell death. The IC50 of the cells were ranged from 3nM to 1.05uM at 72 hours. At 72h, GSK458 10nM lowered cellular mitochondrial potential, ATP levels and glucose uptake. GSK458 induced apoptosis and arrested the cell cycle at G1. At molecular level, GSK458 reduced phosphorylation status of AKT ser473 and Thr308, mTOR and GSK3β. Interestingly, GSK458 inhibited Mcl-1 expression level. GSK458 exhibited synergistic activity when combined with doxorubicin and dexamethasone. To a lesser degree, GSK458 enhanced the anti-tumor activity of Venetoclax, proteasome and HDAc inhibitors. Conclusion: GSK458 is active as a single agent or in combination with chemotherapy agents or small molecule inhibitors in a variety of T-cell pre-clinical models representing forms of systemic or cutaneous T-cell lymphoma. GSK458 was able to inhibit phosphorylation of AKT, mTOR and GSK3β, which may be the mechanism to reduce ATP production and glucose uptake in the cancer cells. Moreover, GSK458 arrested cell cycle at G1 arrest. Our data supports the clinical evaluation of GSK458 in relapsed/refractory T-cell lymphoma patients. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Antibody Drug Conjugate (ADC) Targeting Cell Surface Heat Shock Protein 70 (csHSP70) Is Active Against Pre-Clinical Models of Peripheral T-Cell Lymphoma (PTCL)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 870-870
Author(s):  
Rajan Kumar Choudhary ◽  
Richard J. Jones ◽  
Isere Kuiatse ◽  
Hua Wang ◽  
Francisco Vega ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Rna Seq

Abstract Background: Neoplasms of T-cell or natural killer/T-cell origin account for 10-15% of all non-Hodgkin lymphomas (NHLs) in the United States, and 30% or more of NHLs in African and Asian countries, and tumors from post-thymic or peripheral T-cells are referred to collectively as PTCLs. Recent advances, including approval of brentuximab vedotin (BV), an anti-CD30 monoclonal antibody (mAb) drug conjugate (ADC) with monomethyl auristatin E (MMAE), deacetylase inhibitors (HDACis), and Anaplastic lymphoma kinase (ALK) inhibitors for ALK-positive anaplastic large cell lymphoma (ALCL) have improved outcomes. However, most PTCLs still have a poorer prognosis than comparable B-cell NHLs, and identification of novel targets and drugs retains importance in this area of unmet medical need. Methods: Pre-clinical studies were performed using PTCL and cutaneous T-cell lymphoma (CTCL) cell lines initially in vitro, and then using an in vivo xenograft model. Publically available databases were also leveraged, including the Broad Institute Cancer Cell Line Encyclopedia (CCLE), as well as our own RNA-sequencing (RNA-Seq) data from primary PTCL samples. Results: We examined the cell surface proteome of SUD-HL-1 (ALK+ ALCL), Mac-1 (ALK- ALCL), HH (CTCL), and HuT 78 (Mycosis fungoides with Sézary syndrome) cells by biotinylation and then mass spectrometry, and identified csHSP70 as being consistently expressed in all four lines. Analysis of the CCLE showed that HSP70 mRNA and HSP70 protein was expressed at the highest level in T-cell lymphoma cell lines, and our own RNA-Seq data confirmed HSP70 gene expression was higher in primary PTCL samples, and especially in ALCLs, compared with normal T-cells. To test its promise as a therapeutic target, we generated mAbs to human HSP70 and identified one clone, 239-87, which specifically bound csHSP70 on T-cell NHL cell lines but not on normal peripheral blood-derived mononuclear cells (PBMCs). Next, 239-87 was linked to MMAE to generate an ADC with a drug:antibody ratio of 4, and we confirmed that it was both internalized and then trafficked into acidic vacuoles in SUD-HL-1 cells. The 239-87-MMAE ADC induced a time- and concentration-dependent loss of viability in a panel of PTCL and CTCL cell lines associated with a G2/M arrest and induction of apoptosis, while normal PBMCs were unaffected. Comparisons of the activity of BV with 239-87-MMAE showed that the latter had similar efficacy against SU-DHL-1 and Hut 78 cells in vitro. When cells were propagated under conditions of hypoxia to mimic the tumor microenvironment there was an increase in csHSP70 expression, and the sensitivity of PTCL and CTCL cell lines to the 239-87-MMAE ADC was enhanced. Conversely, an inducible HSP70-targeted short hairpin RNA reduced total and csHSP70 protein expression, and reduced the efficacy of the ADC. Also of note, the HDACi vorinostat enhanced csHSP70 levels, and combinations of vorinostat with the 239-87-MMAE ADC enhanced loss of viability in these cells in a synergistic manner based on combination index analyses. Finally, we prepared an orthotopic in vivo PTCL model by subcutaneously injecting luciferase-labeled Mac-1 cells into C.B-17/IcrHsd-Prkdc scid mice. Disease progression occurred rapidly in all mice treated once weekly on days 10, 17, 24, and 31 with an IgG2A isotype mAb, as was the case for 7/8 mice treated with the 239-87-MMAE ADC at 1 mg/kg. In contrast, palpable tumor disappeared in 1/8 mice that received this ADC at 1 mg/kg, and 8/8 and 7/7 mice that received dosing at 5 and 10 mg/kg, respectively (Figure 1A). Tumor recurrence has not been seen at 105 days, including 74 days since the last ADC dose, and the one mouse at 1 mg/kg, and 3 each in the 5 and 10 mg/kg cohorts have had no disease by imaging, while the others have a small residual signal (Figure 1B) that has not progressed for two months. Conclusions: These pre-clinical in vitro and in vivo data support the possibility that csHSP70 could represent a novel therapeutic target for PTCL, and provide a rationale to translate ADCs based on our clone 239-87 mAb to the clinic for patients with advanced ALCL, and potentially other T-cell lymphomas as well. Figure 1 Figure 1. Disclosures Jones: Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company. Vega: i3Health, Elsevier, America Registry of Pathology, Congressionally Directed Medical Research Program, and the Society of Hematology Oncology: Research Funding; CRISPR Therapeutics and Geron: Research Funding. Orlowski: Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria; CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Functional Characterization of BCL2-Family Members Identifies BH3 Mimetics As Potential Therapeutics in T-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 292-292 ◽  
Author(s):  
Raphael Koch ◽  
Elizabeth Brem ◽  
Rachael Clark ◽  
Thomas S. Kupper ◽  
Anthony Letai ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Family Members ◽  
T Cell Lymphoma ◽  
Bh3 Mimetics ◽  
T Cell Lymphomas ◽  
Pdx Models

Abstract Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of lymphoid malignancies with generally poor outcomes when treated with current regimens. The pro-survival BCL-2 family members BCL-2, BCL-xL, and MCL-1 contribute to tumor maintenance, progression, and chemoresistance across a range of cancers but their contributions in distinct subtypes of PTCL are poorly understood. Immunohistochemical analyses of PTCL specimens have revealed a distinct expression pattern of BCL-2 family members, most notably the high level expression of BCL-2 in up to 50% of certain PTCL entities (Rassidakis et al., J Pathol 2003). In fact, high BCL-2 expression has been associated with unfavorable prognosis (Ling et al., Biomed Environ Sci 2011). We amassed a collection of 21 T-cell lymphoma cell lines (representing Alk+ ALCL, Alk- ALCL, PTCL-NOS, cutaneous T-cell lymphoma (CTCL) and rare subtypes) and 7 patient-derived xenograft (PDX) models of T-cell lymphoma. The latter include models of Alk+ ALCL, Alk- ALCL, ATLL, NK-T cell lymphoma and AITL (available at http://www.PRoXe.org) (Townsend et al. Cancer Cell 2016). To assess the expression level and protein abundance of BCL2 family members, we performed RNA-Seq and immunoblotting. To functionally characterize dependence on BCL-2 family members, we utilized BH3 profiling, a technique that allows for assessment for how "primed" or close to the cell death threshold cells are by evaluating the degree of mitochondrial outer membrane permeabilization (MOMP), induced by a panel of BH3 domain peptides (Ryan and Letai, Cell Death and Differentiation 2013). Binding specificity of BH3 domain peptides allows for determination of which pro-survival Bcl-2 family members cells are dependent on for survival and thus makes it a powerful tool to predict response to BH3 mimetics. Finally, we assessed in vitro the cytotoxicity induced by the BH3 mimetics venetocxlax (ABT-199, a BCL-2 specific agent) and navitoclax (ABT-263, which targets both BCL-2 and BCL-xL) in PTCL cell lines. Gene expression and protein levels of the anti-apoptotic BCL-2 family members showed a distinct pattern in the cell lines that closely recapitulated immunohistochemical analysis of clinical samples (Rassidakis et al., J Pathol 2003). Specifically, both MCL-1 and BCL-xL were ubiquitously expressed, with higher levels of MCL-1 in ALCL cell lines and the PTCL-NOS cell line SMZ-1, while BCL-xL was highly expressed predominately in CTCL cell lines. While cell lines and PDX models from Alk+ ALCL and CTCL universally did not express BCL-2, approximately two-thirds of cell lines and PDX models representing Alk- ALCL, PTCL-NOS, AITL, NK/T-cell lymphoma, ATLL and rare subtypes of T-cell lymphomas did express BCL-2. Despite this expression, only 3 of 8 BCL2-expressing cell lines were sensitive to ABT-199 (IC50<1 µM), indicating that BCL2 expression is an inadequate biomarker for ABT-199 sensitivity. In contrast, BH3 profiling of these models identified either exclusive BCL-2 dependence, which correlated with sensitivity to ABT-199 in vitro, or exclusive MCL-1 dependence, which correlated with resistance to ABT-199. Alk+ and Alk- ALCL cell lines and PDX models were predominately MCL-1 dependent, but some also showed co-dependence on BCL-xL that correlated with sensitivity to ABT-263 in vitro. Among CTCL cell lines, we identified a dominant BCL-xL dependence that correlated with low nanomolar IC50 to ABT-263. In line with this, primary samples of CTCL (n=3) also showed BCL-xL dependence, offering a novel therapeutic strategy for this disease. Table 1 shows a representative illustration of these data in a selection of cell lines. In summary, we have defined distinct classes of BCL-2 family member dependence that are revealed by BH3 profiling and predict sensitivity or resistance to available clinical agents. Disclosures Letai: AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Weinstock:Novartis: Consultancy, Research Funding.

Results From a Prospective, Open-Label, Phase II Trial of Bendamustine in Refractory or Relapsed T-Cell Lymphomas: The BENTLY Trial

2013 ◽  
Vol 31 (1) ◽  
pp. 104-110 ◽  
Author(s):  
Gandhi Damaj ◽  
Rémy Gressin ◽  
Krimo Bouabdallah ◽  
Guillaume Cartron ◽  
Bachra Choufi ◽  
...  
Keyword(s):  
T Cell ◽  
Response Rate ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Open Label ◽  
Previous Response ◽  
Angioimmunoblastic Lymphadenopathy ◽  
Prior Chemotherapy ◽  
T Cell Lymphomas

Purpose To determine the efficacy and safety of bendamustine as a single agent in refractory or relapsed T-cell lymphomas. Patients and Methods Patients with histologically confirmed peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma who progressed after one or more lines of prior chemotherapy received bendamustine at 120 mg/m2 per day on days 1 through 2 every 3 weeks for six cycles. The primary end point was overall response rate (ORR). Secondary end points were duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results Of the 60 patients included, 27 (45%) were refractory to their last prior chemotherapy, and the median duration of the best previous response was 6.6 months. Histology was predominantly angioimmunoblastic lymphadenopathy and PTCL not otherwise specified. The disease was disseminated in the majority of patients (87%). The median number of previous lines of chemotherapy was one (range, one to three). Twenty patients (33%) received fewer than three cycles of bendamustine, mostly because of disease progression. In the intent-to-treat population, the ORR was 50%, including complete response in 17 patients (28%) and partial response in 13 patients (22%). Bendamustine showed consistent efficacy independent of major disease characteristics. The median values for DoR, PFS, and OS were 3.5, 3.6, and 6.2 months, respectively. The most frequent grade 3 to 4 adverse events were neutropenia (30%), thrombocytopenia (24%), and infections (20%). Conclusion Bendamustine showed an encouraging high response rate across the two major PTCL subtypes, independent of age and prior treatment, with acceptable toxicity in refractory or relapsed T-cell lymphoma.

Pralatrexate Is Active in Cutaneous T-Cell Lymphoma (CTCL): Results of a Multicenter, Dose-Finding Trial.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 919-919
Author(s):  
Steven M. Horwitz ◽  
Madeleine Duvic ◽  
Youn Kim ◽  
Jasmine M Zain ◽  
Mary Jo Lechowicz ◽  
...  
Keyword(s):  
T Cell ◽  
Adverse Events ◽  
Dose Reduction ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Phase 1 ◽  
Single Agent ◽  
Optimal Dose ◽  
T Cell Lymphoma ◽  
Grade 3

Abstract Abstract 919 Background: Pralatrexate enters cancer cells via the reduced folate carrier-1 (RFC-1) and is efficiently polyglutamated by folylpolyglutamyl synthetase (FPGS), leading to high intracellular retention. In a Phase 1/2 study of patients with hematologic malignancies, pralatrexate demonstrated activity in aggressive T-cell lymphoma with a maximum tolerated dose (MTD) of 30 mg/m2 once weekly for 6 of 7 weeks. The generally indolent course of CTCL may be better treated at lower doses in a maintenance fashion if a lower incidence and severity of adverse events can be achieved while preserving activity. PDX-010 is an open-label, single-agent, multicenter, Phase 1 dose-reduction trial in patients with relapsed or refractory CTCL. The primary objective is to identify an optimal dose and schedule of pralatrexate for these patients. Methods: Eligibility included mycosis fungoides (MF), Sézary syndrome (SS), and primary cutaneous anaplastic large cell lymphoma (ALCL); with disease progression after at least 1 prior systemic therapy. The pralatrexate dose and schedule started at 30 mg/m2 by IV push on 3 of 4 weeks and subsequent cohorts received reduced doses (20, 15, 10 mg/m2) and/or schedules (3/4 or 2/3 weeks) of pralatrexate based on tolerability. All patients received supplementation with vitamin B12 1 mg intramuscularly every 8-10 weeks and folic acid 1 mg orally once daily. As we sought a well tolerated regimen the definition of DLTs to trigger dose reduction included toxicities such as grade ≥ 3 neutropenia, grade ≥ 2 thrombocytopenia, febrile neutropenia, grade ≥ 2 mucositis/stomatitis, and any toxicity leading to dose omission or reduction in cycle 1. If DLT occurred and a response was seen, the following cohort was opened at the next lower dose or next less frequent schedule. Response was evaluated by modified severity-weighted adjustment tool (SWAT) every 2 cycles for 6 months and then every 4 cycles. For patients with lymph node involvement, scans were completed at baseline and upon clinical response or end of treatment, whichever occurred first. Results: Thirty-one patients received pralatrexate, with 18 (58%) men and median age of 57 yrs (range, 30-81). Patients had received a median of 6 prior therapies (range, 1-25). Cohorts at the following doses/schedules were enrolled: 30 mg/m2 x 3/4 weeks (n=2), 20 mg/m2 x 3/4 weeks (n=3), 20 mg/m2 x 2/3 weeks (n=7), 15 mg/m2 x 3/4 weeks (n=6), 15 mg/m2 x 2/3 weeks (n=3), and 10 mg/m2 x 3/4 weeks (n=10). Patients received pralatrexate for a median of 72 days (range, 7-491+); 4 patients received >10 cycles of treatment. The most common treatment-related adverse events (all grades) were mucositis (18 patients [58%]), nausea (14 patients [45%]), fatigue (14 patients [45%]), pyrexia (7 patients [23%]), vomiting (6 patients [19%]), anemia (6 patients [19%]), and edema (5 patients [16%]). Grade 3-4 treatment-related toxicities in >1 patient each were mucositis (4 patients [13%]) and anemia (2 patients [6%]). Mucositis was dose limiting (≥ grade 2) in 8 patients (26%). A total of 11 responses were observed, including 2 complete responses and 9 partial responses. In the 18 patients who received pralatrexate at a dose intensity of 15 mg/m2 x 3/4 weeks or greater, the objective response rate was 56% (10/18 patients). This appeared to be the threshold dose for substantial activity in CTCL, below which the incidence of responses decreased in this dose de-escalation trial. Conclusion: Pralatrexate shows impressive activity in the treatment of relapsed CTCL. The optimal dose and schedule that provided activity with tolerability for CTCL was determined to be pralatrexate 15 mg/m2 weekly on 3 of 4 weeks. This cohort is being expanded to better assess efficacy and durability. Disclosures: Horwitz: Allos Therapeutics, Inc: Consultancy, Research Funding. Duvic:Allos Therapeutics, Inc.: Research Funding. Lechowicz:Allos Therapeutics, Inc.: Consultancy. Fruchtman:Allos Therapeutics, Inc.: Employment.

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