Mimetic Peptide to CDK Inhibitor p16 INK4a Induces Cell Death In Mantle Cell Lymphoma Cells: A New Strategy to Eradicate Minimal Residual Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3921-3921
Author(s):  
Hongtao Liu ◽  
Greg Koval ◽  
Greg Malnassy ◽  
Pamela Ihonor ◽  
Hui Liu ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Mantle Cell Lymphoma ◽  
Residual Disease ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Induce Cell Death ◽  
Mimetic Peptide ◽  
Mantle Cell ◽  
Minimal Residual

Abstract Abstract 3921 Autologous stem cell transplantation (ASCT) is an effective treatment strategy for Mantle Cell Lymphoma (MCL) that has been shown to improve disease free survival. However, data from recent trials suggest that the presence of minimal residual disease (MRD) contributes to relapse following current intensive treatment strategies, including ASCT. Thus, we sought to test a highly selective approach to eradication of MRD in stem cell collections from MCL patients using targeted small peptides that disrupt the aberrant cyclin/CDKs interactions that are involved in the pathogenesis of MCL. In the current study, the efficacy of a mimetic peptide to the CDK inhibitor p16INK4a to eliminate MRD was tested. Using (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) cell viability assays, we demonstrated that a transducible TAT-p16 mimetic peptide was able to induce cell death in the MCL cell line, Jeko-1, in a dose and time-dependent fashion (see Figure). A TAT-p16 mimetic was able to induce cell death in approximately 60% of Jeko-1 cells after 4 hours of incubation, and approximately 80% of Jeko-1 cells at 24 hours. In contrast, the same dose of TAT-p16 mimetic had no effect on cell survival when it was incubated with CD34+ rich apheresis samples from healthy donors. Furthermore, CFU-GM, BFU-E colony assays showed that there was no toxicity of the TAT-p16 mimetic when compared to untreated cells or cells treated with a control, scrambled TAT-p16 peptide. TAT-p16 mimetic did not impair erythroblast differentiation but induced mild apoptotic cell death in the erythroblast cells. The strategy was further tested using a CD34+ enriched apheresis sample collected from a patient with MCL with documented MRD who had been treated on a clinical trial of ASCT for MCL. Using Real-time quantitative PCR for IgH and BCL1 gene rearrangements, we demonstrated that the TAT-p16 mimetic was able to reduce MRD level by 40% compared with the scrambled TAT-p16 peptide. These data suggest that the use of a peptide-mimetic to p16 selectively and effectively can reduce MRD in MCL cells and in an apheresis (stem cell) sample from a patient with MCL. Treatment with the peptide allowed differentiation of CD34+ progenitor cells. A combinational approach using additional targeted cell cycle regulatory peptides that block p21CIP1/WAF1 and cyclin D2 is being explored to optimize the efficacy of this purging technique. Thus, this novel strategy may be an effective, selective and non-toxic method for eradication of MRD in MCL. Disclosures: No relevant conflicts of interest to declare.

The HDACi Romidepsin Markedly Synergizes With Inhibition Of ATM By KU60019 In Mantle Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3066-3066 ◽  
Author(s):  
Luigi Scotto ◽  
Kelly Zullo ◽  
Xavier Jirau Serrano ◽  
Laura K Fogli ◽  
Owen A. O'Connor
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a disease characterized by gross cell cycle dysregulation driven by the constitutive overexpression of cyclin D1. The identification of a “proliferation signature” in MCL, underscores the necessity of new therapeutic approaches aimed at lowering the proliferative signature of the disease, theoretically shifting the prognostic features of the disease. Romidepsin, an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. However up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDACi activates p21Cip1/Waf1 expression via ATM and KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the effect of the combination of romidepsin and KU60019 in inducing cell death in MCL. Analysis of romidepsin treated Jeko-1 cell extracts showed a marked effect on the expression of proteins involved in cell cycle regulation. Decrease expression of Emi1, a mitotic regulator required for the accumulation of the APC/C substrates was observed. Emi1 is also responsible for the stability of the E3 ubiquitin ligase Skp2 that specifically recognizes and promotes the degradation of phosphorylated cdk inhibitor p27. However, decrease in Emi1 protein levels, upon addition of romidepsin, was not followed by an increased expression of the cdk inhibitor p27. On the other end, increased expression of the cdk inhibitor p21Cip1/Waf1, was a common feature of all romidepsin treated MCL lines analyzed. Cell cycle analysis via Fluorescent Activated Cell Sorting (FACS) of romidepsin treated Jeko-1 cells showed an accumulation of romidepsin treated cells in the G2/M phase when compared to the control suggesting a p21Cip1/Waf1 induced cell cycle arrest. For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM followed by acquisition on a Biotek Synergy HT and IC50s calculated using the Calcusyn software. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR). Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells treated with different concentrations of romidepsin corresponding to IC10-20 in combination with KU60019 at a concentration of 2.5, 5.0, 7.5 and 15 umol/L for 24, 48 and 72 hours. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all concentrations. The RRR analysis showed a strong synergism at 48 and 72 hours in virtually all combinations of HDACi and KU60019 in all three cell lines. The results of drug:drug combination in two of the three cell lines are shown below. Protein expression analysis of Jeko-1 and Maver-1cells treated with single agents or combinations for 48 hours revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. The increased p21 protein expression upon addition of romidepsin, was not observed when the romidepsin treatment was combined with the KU60019. Increased activation of the programmed cell death proteins Caspase 8, induced by Fas, and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1). Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with romidepsin plus increase concentrations of KU60019 when compared with their abundance in the presence of the single agents. Cell cycle analysis of Jeko-1 cells treated for 24 hours with single agents and combination suggests that the increased apoptosis is the result of inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest by KU60019. Overall, these data demonstrated that the combination of romidepsin and KU60019 was synergistically effective in inhibiting the in vitro growth of the mantle cell lymphoma lines. Jeko-1 Maver-1 Disclosures: O'Connor: Celgene: Consultancy, Research Funding.

scholarly journals Minimal Residual Disease Monitoring By 8-Color Flow Cytometry in Mantle Cell Lymphoma Is Complementary to Q-PCR Monitoring and Will Facilitate Pre-Emptive Treatment: An EU-MCL and Lysa Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1657-1657
Author(s):  
Morgane Cheminant ◽  
Stephanie Schmit ◽  
Aurore Touzart ◽  
Coralie Derrieux ◽  
Marie-Hélène Delfau-Larue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mantle Cell Lymphoma ◽  
Patient Management ◽  
Residual Disease ◽  
Cell Lymphoma ◽  
Clinical Relapse ◽  
Color Flow ◽  
Mantle Cell ◽  
Minimal Residual

Abstract Introduction: Mantle Cell Lymphoma (MCL) is characterized by frequent blood and bone marrow involvement. It has been demonstrated that use of Minimal Residual Disease (MRD) quantification in blood and/or bone marrow might be helpful in patient management. Gold standard MRD is based on Q-PCR clone specific amplification of IgH VDJ or IgH-BCL1 rearrangements, but these are relatively complex and time consuming and over half of the positive results are in a grey zone of borderline positivity. Flow cytometry (FCM) is more rapid and better adapted to individual patient management if quantitatively reproducible, but insufficiently sensitive when only 4 colors are used. We therefore developed a universal, 8-color, EuroFlow inspired, FCM strategy, which we compared with classical Q-PCR MRD in 61/97 patients included in (and 1 treated according to) the EU-MCL Younger and Elderly prospective trials who underwent Q-PCR MRD monitoring at Necker Hospital. Method: Q-PCR MRD from IgH VDJ (n=92) or BCL1-IgH (n=5) was performed prospectively from ficolled blood (PB) or bone marrow, from which residual material was cryopreserved in DMSO for FCM quantitation, using 10 antibodies labelled with 8 fluorochromes for positive and negative (CD45, CD19, CD5, LAIR1, CD11a, IGK, IGL, CD3, CD14 and CD56) gating, after diagnostic phenotyping of fresh material, using the same panel and a EuroFlow B lymphoid screening tube. Sensitivity of both techniques was at least 0.01% (1E-04). FCM was only considered positive if above 0.01%, whereas Q-PCR results were considered positive below quantifiable range (BQR) if borderline, above sensitivity, within Euro-MRD criteria for MRD positivity. BQR samples were separated based on the number of positive, triplicate samples. The objectives were to compare the two techniques and to determine their suitability for regular screening, with a view to pre-emptive treatment on molecular or phenotypic (MRD) relapse. Two patients were treated with Rituximab at MRD relapse, prior to clinical relapse, as proof of principle. Results: A total of 302 blood or bone marrow samples from 62 patients were quantified. Overall, 79% (42/53) of samples positive at or above 0.01% by PCR were also positive by FCM, compared to 29% (19/65) of those below 0.01%, but with at least 2 positive triplicates and virtually none of those with only 1 or no results above sensitivity (1%, 2/184). Quantification of the paired MRD results positive with PCR and/or FCM were significantly correlated (r2=0.74, P<0.0001). Amongst the 62 patients, 30 have relapsed and 19 have died. Nine relapsing patients (including one off protocol patient treated and monitored at initial and second MRD relapses) had sufficient MRD points to assess the capacity of PB Q-PCR or FCM to predict future clinical relapse sufficiently to justify pre-emptive treatment at MRD relapse. Clinical relapse was preceded by MRD relapse in 9/10 relapses by Q-PCR and 7/9 by FCM. Six of the 9 relapsing patients had achieved Q-PCR negativity in at least one PB sample. The mean latency for prediction by Q-PCR, when considering any increase in positivity to at least 2 positive triplicates as positive, was 11.3 months (range 1-24mths) and 5.4 months (0.5-11) when only results above 0.01% were considered positive. The equivalent latency by FCM was slightly shorter, at 6.5 months (0.5-21) Pre-emptive treatment of 2 patients at MRD relapse, prior to clinical relapse allowed re-establishment of molecular complete remission and a durable second remission in at least one with sufficient follow-up (Cf Fig.). Figure 1 Figure 1. Conclusion: Eight color flow cytometry is a promising alternative to classical clone-specific Q-PCR strategies in monitoring therapy in MCL, with an excellent correlation (29/31, 94%) for MRD levels of at least 0.1% and acceptable correlation at 0.01-0.1% (13/22, 59%). While less sensitive at very low levels on cryopreserved material, FCM may clarify the clinical relevance of low-level borderline positivity; however it remains to be determined prospectively which technique will have greater prognostic value in patient management. FCM sensitivity will be improved by prognostic testing of fresh whole blood or bone marrow, and this pilot data clearly justifies such studies. Finally, MRD relapse precedes clinical relapse by several months, justifying pre-emptive treatment, monitored by prospective FCM and IgH Q-PCR within clinical trials. Disclosures Dreyling: Roche: Honoraria, Research Funding.

scholarly journals Ofatumumab Plus Hypercvad/MA Induction Leads to High Rates of Minimal Residual Disease (MRD) Negativity in Patients with Newly Diagnosed Mantle Cell Lymphoma, Results of a Phase 2 Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2817-2817
Author(s):  
Pallawi Torka ◽  
Nishitha Reddy ◽  
Adrienne Groman ◽  
Angela Kader ◽  
Jenna Nichols ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Mantle Cell Lymphoma ◽  
Research Funding ◽  
Residual Disease ◽  
Cell Lymphoma ◽  
Survival Outcomes ◽  
Phase 2 ◽  
Newly Diagnosed ◽  
Mantle Cell ◽  
Minimal Residual

Background: Minimal residual disease (MRD) status is an independent prognostic marker for response duration in patients with mantle cell lymphoma (MCL). Rituximab-based therapy led to a molecular remission (MR) in 48% young MCL patients treated with intense chemo-immunotherapy. More recently, 4 courses of R-DHAP were reported to yield a 66% MRD negativity rate prior to high dose chemotherapy and autologous stem cell transplant (HDC-ASCT). Ofatumumab is a humanized type 1 anti-CD20 mAb that binds to a unique, more membrane-proximal epitope on the CD20 antigen. Pre-clinical studies have demonstrated that ofatumumab (O) is more active than rituximab in eliciting complement-mediated cytotoxicity (CMC) in MCL cell lines, primary tumor cells and murine lymphoma xenograft models. Hence, we evaluated the safety and efficacy of combining ofatumumab with HyperCVAD/MA (O-HyperCVAD) in newly diagnosed MCL (NCT01527149). Study design: This was a single-arm, open-label, multi-center (2 centers), prospective, phase 2 clinical trial. Thirty-seven transplant-eligible patients with newly diagnosed MCL were enrolled. Ofatumumab (1000mg) was given every 3 weeks on day 1, followed by standard doses of alternating HyperCVAD and MA starting on day 3. A total of 6 cycles were given at 3-week intervals followed by HDC-ASCT. Maintenance rituximab 375 mg/m2 every 2 months for 3 years post-ASCT was added after protocol amendment following publication of the LyMa study. Primary objectives were to determine the overall response rate (ORR) and CR rate (CRR) at the end of therapy. Secondary objectives included MRD negativity, progression free survival (PFS), overall survival (OS), feasibility of successful mobilization of autologous stem cells and safety assessment. MRD assessment was performed in peripheral blood (PB) and bone marrow (BM) using high sensitivity multiparametric flowcytometry at baseline, after 2 cycles, after 4 cycles, pre-ASCT, post-ASCT and 6 months post-ASCT. Exploratory endpoints included correlation of MRD with survival, correlation of surface CD20 levels with response and to compare differences in ORR according to Cheson and modified Cheson (MC) criteria. Results: Median age was 60yrs; 73% of patients were male, 92% had an ECOG PS 0-1, majority (81%) had stage 4 disease with 86% having bone marrow involvement; 22% had low risk, 43% had intermediate risk and 35% had high risk MIPI score; 11% had blastoid and 11% had pleiomorphic variants of MCL; 25% harbored p53 deletion. MRD was assessed in 28 of 37 patients; 9 patients from partner site were excluded due to time lag in sample delivery for flowcytometry. MRD positivity was noted in 75% patients in PB and 71.4% patients in BM at baseline. Subsequent samples showed an MRD negativity rate of 82.1% and 64.3% after 2 cycles and 76% and 96% pre-ASCT in PB and BM respectively. Post-ASCT, MRD in BM and PB was found negative in 58% and 90%, respectively. Attainment of early MRD negativity (after 2 cycles) was associated with improved PFS (p=0.04) and OS (p=0.03). Since majority of patients were MRD negative pre-ASCT, we could not demonstrate correlation between pre-ASCT MRD and survival outcomes. The ORR and CRR were 95% and 62% by Cheson and 97% and 84% respectively by modified Cheson criteria. At the end of induction therapy, ORR was 86% and CRR was 62% by Cheson criteria. 73% pts underwent HDC-ASCT. Only one patient failed stem cell collection. The median PFS and OS were 45.5 months and 56 months respectively. There were 3 deaths while on therapy- 2 from sepsis and one from acute myeloid leukemia. Grade 3 and 4 adverse events (AEs) were observed in 22% and 68% of the patients, most of them were hematological AEs related to the chemotherapy regimen. Correlation of surface CD20 expression using mean fluorescent intensity (MFI) and response rates is currently under analysis and will be reported at the meeting. Conclusion: The addition of ofatumumab to HyperCVAD/MA led to high rates of MRD negativity by flowcytometry in patients with newly diagnosed MCL. Early MRD negativity (after 2 cycles) was associated with better PFS and OS. Despite higher ORR, CR and MRD negativity rates, survival outcomes were similar to historical cohorts using rituximab-HypeCVAD/MA. This may be in part due to increased number of patients with high-risk disease in our study. Disclosures Reddy: KITE Pharma: Consultancy; Abbvie: Consultancy; Genentech: Research Funding; Celgene: Consultancy; BMS: Consultancy, Research Funding. Sait:Celgene: Consultancy. OffLabel Disclosure: Ofatumumab was investigated in combination with chemotherapy in newly diagnosed mantle cell lymphoma in this clinical trial.

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