Experts from the NYU Cancer Institute , an NCI-designated cancer center at NYU Langone Medical Center , presented their latest research findings about hematologic cancers at the 53rd ASH Annual Meeting and Exposition held December 10-13, 2011 in San Diego, California. Significant research news shared about lymphomas, Acute Lymphoblastic Leukemia (ALL) and multiple myeloma by NYU Cancer Institute experts included:
Reinduction Chemoimmunotherapy with Epratuzumab in Relapsed Acute Lymphoblastic Leukemia (ALL) in Children, Adolescents and Young Adults: Results From Children’s Oncology Group (COG) Study ADVL04P2
Oral Abstract 573: Monday, December 12, 2011, 3:15PM
Authors:Elizabeth A. Raetz, MD, and William L. Carroll, MD, NYU Cancer Institute
Outcomes for children with relapsed Acute Lymphoblastic Leukemia(ALL) remain poor, especially when relapses occur early less than 36 months following initial diagnosis. A factor contributing to poor outcomes is much lower rates of successful induction of second remission (CR2). CD22 is almost universally expressed in children with B-cell precursor (BCP) ALL and we previously demonstrated the safety of adding the anti-CD22 monoclonal antibody, epratuzumab, to reinduction chemotherapy in children with first marrow relapse. The primary aim of the second part of the COG ADVL04P2 study was to determine if addition of epratuzumab to an established chemotherapy platform improves rates of CR2 in individuals with BCP ALL and early bone marrow relapse. This report focuses on ADVL04P2 part B and includes results of both the weekly (B1) and twice weekly (B2) epratuzumab dosing schedules. At the end of block 1, 48 B1 patients and 50 B2 patients were evaluable for response with CR2 achieved in (31/48) 65% of B1 and (33/50) 66% of B2 patients. Minimal residual disease (MRD) was measured by flow cytometry in a COG reference laboratory at the end of block 1. Epratuzumab, as given on the B1 and B2 schedules was tolerable in combination with chemotherapy in pediatric and young adult patients with early relapsed CD22-positive BCP ALL, but did not improve CR2 rates when compared to historical controls treated with chemotherapy alone. However, among patients who attained a complete remission, those treated with epratuzumab were significantly more likely to become MRD negative as compared to those treated without epratuzumab.
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Epigenetic Modulation Leads to Re-Expression of Relapse Specific Silenced Genes and Induces Chemosensitivity in Childhood Acute Lymphoblastic Leukemia (ALL)
Poster Abstract 3455: Monday, December 12, 2011, 6:00PM
Authors: Teena Bhatla, MD and William L. Carroll, MD, NYU Cancer Institute
The poor outcome of patients with Acute Lymphoblastic Leukemia (ALL) who relapse and the frequent failure of conventional salvage strategies mandate novel treatment approaches. Previous work NYU Cancer Institute laboratory led to the discovery that the histone deacetylase inhibitor (HDACi) vorinostat could effectively reverse the expression of a relapse-specific gene signature and restore blast chemosensitivity in vitro. We now have also performed genome wide DNA methylation profiling in 33 matched diagnosis/relapsed paired marrow samples by Infinium Human Methylation27 Beadchip array obtained from patients enrolled on Children’s Oncology Group (COG) protocols and observed that blasts at relapse are significantly more methylated compared those at diagnosis (p<0.00001) with concordant downregulation of gene expression. Authors conclude, decitabine leads to re-expression of genes shown to be preferentially methylated at relapse and is synergistic when applied prior to conventional chemotherapy. Combination therapy with vorinostat results in maximal cell kill in cell lines and primary patient samples. Targeted epigenetic therapy with agents such as decitabine and vorinostat may be a promising approach to the treatment of relapsed ALL.
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Hodgkin’s Lymphoma Cell Lines Have up-Regulated IL-3 Receptor α (IL-3Rα) Expression and Are Sensitive to SL-401, An IL-3Rα Targeted Drug
Poster Abstract 3737: Monday, December 12, 2011, 6:00PM
Authors: Catherine S. Diefenbach, MD and Owen A. O'Connor, MD, PhD, NYU Cancer Institute
Hodgkin lymphoma (HL) is among the most curable lymphomas, however 20%-30% of patients relapse after initial chemotherapy, or have primary refractory disease. While 30-50% of these patients may be cured with second line chemotherapy and autologous stem cell transplant, patients who do not obtain a complete response (CR) prior to transplantation, or who relapse after second line therapy have few effective therapeutic options. Novel treatment strategies for these patients are needed. It has been previously shown that in patients with classical HL the malignant Hodgkin Reed-Sternberg cells (HRS) express a high level of the Interleukin-3 receptor (IL-3R). Therefore, we hypothesized that SL-401, a novel biologic conjugate consisting of IL-3 linked to diphtheria toxin, which targets IL-3R, may be an effective approach for selectively targeting and killing HRS cells. Study results suggest that CD123 expression may vary as a function of HL histology, and that sensitivity to SL-401 may correlate with CD123 expression. Based on these results, SL-401, which is currently being evaluated in clinical trials of patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myeloid leukemia, may be a potential treatment strategy in refractory HL, and warrants further exploration in T-ALL. Exploration of CD123 expression in primary and relapsed/refractory patients with lymphoid malignancies as well as in vivo studies with SL-401 in this setting is currently underway.
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Combination of Epigenetic Agents Synergistically Reverse the Malignant Phenotype in Models of T-Cell Lymphoma
Poster Abstract 2727: Sunday, December 11, 2011, 6:00PM
Authors: Enrica Marchi, MD, PhD and Owen A. O'Connor, MD, PhD, NYU Cancer Institute
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). 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.
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Preliminary Results of An Ongoing Phase I Trial of Oral Belinostat a Novel Histone Deacetylase Inhibitor in Patients with Lymphoid Malignancies
Poster Abstract 3710: Monday, December 12, 2011, 6:00PM
Authors: Jasmine M. Zain, MD and Owen A. O'Connor, MD, PhD, NYU Cancer Institute
Belinostat (Bel) is a pan class I/II histone deacetylase inhibitor with broad preclinical activity. A phase I study of oral Bel in patients (pts) with solid tumors identified a maximum tolerated dose (MTD) of 750 mg orally (PO) daily on days (d) 1-14, of an every 21 day cycle. An allowance for intra-patient dose escalation was permitted as long as the higher dose level was deemed safe and not the maximum administrable dose. The current study was initiated to assess the safety and dosing of Bel in patients with relapsed or refractory Hodgkin and non-Hodgkin Lymphoma. 28 pts, median age 48 (range 21-82),prior regimens: median 5,5 (range 0-13) , (17 had BM transplants, including 5 pts with allogeneic) have been enrolled. Authors concluded, Oral Bel can be delivered safely with a d 1-14, q3w schedule in patients with lymphoma at a daily dose higher than what has been established for patients with solid tumors. MTD for lymphoma pts was established at 1500 mg/d 1-14, q3w. The safety profile and early tumor shrinkage noted in both HD, MCL and other NHL warrants continued evaluation of Bel, especially in combination with other active compounds.
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Therapeutic Targeting of the Bcl-6: p53 Axis with Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma
Poster Abstract 3733: Monday, December 12, 2011, 6:00PM
Authors: Jennifer E. Amengual, MD and Owen A. O'Connor, MD, PhD, NYU Cancer Institute
Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's Lymphoma. Despite advances in treatment, 1/3 of patients die from their disease. Gene expression profiling has delineated three subtypes with different genetic features known to be prognostic: the Activated B-cell (ABC), Germinal Center (GC), and grey zone types. For example, ABC DLBCL is addicted to NFkB over-expression. The oncogene, BCL6, encodes a transcription factor that functions as a transcriptional repressor within normal germinal center B-cells. Constitutive activation of Bcl-6 leads to GC-type DLBCL by turning off genes expressing cell cycle dependent kinase inhibitors, and essential tumor suppressor genes, like p53. There is a critical inverse relationship between Bcl-6 and p53, the functional status of which is linked to each transcription factor's degree of acetylation. Deacetylation of Bcl-6 is required for maintaining its effects as a transcriptional repressor. Conversely, acetylation of p53 is activating when class III histone deacetylases (HDAC), also known as sirtuins, are inhibited by drugs such as niacinamide. HDAC inhibitors are presently approved for T-cell lymphoma and may require the targeting of additional pathways to be effective in B-cell lymphomas. Trichostatin A and niacinamide modulate Bcl-6 in lymphoma cell lines. One therapeutic strategy that could favorably shift the relationship between oncogenes and tumor suppressors is the pharmacologic modification of Bcl-6 and p53 using HDAC inhibitors. Eight DLBCL cell lines were screened with four class I/II HDAC inhibitors (romidepsin, vorinostat, panobinostat and belinostat) in combination with niacinamide (sirtuin inhibitor). Cell growth inhibition was measured by luminescence cell viability and apoptosis flow cytometry assays. Synergy was achieved in significantly greater number and intensity in the GC versus ABC cell lines. Specifically, romidepsin in combination with niacinamide achieved the greatest synergy. To analyze mechanism of action, DLBCL cell lines were treated with combinations of class I/II HDAC inhibitors and niacinamide. Cells of both GC and ABC subtypes treated with the combination resulted in increased acetylation of p53, and increased p21 and BLIMP-1 content compared to controls. These results did not correlate with cytotoxicity as the ABC cell lines did not achieve the same synergy as the GC cells. GC cells treated with the same combinations resulted in acetylation of Bcl-6 compared with controls as measured by immunoprecipitation and Western blotting assays; ABC cells do not express Bcl-6. This finding correlated with cytotoxicity implying that a rational second pathway must be targeted to shift the balance between oncogene and tumor suppressor activity to achieve effective cell kill. p300 content was also increased suggesting that treatment with HDAC inhibitors recruit or upregulate its production and activity leading to increased acetylation. Using a novel double transgenic mouse model of aggressive spontaneous B-cell lymphoma (l-myc overexpressing crossed with CD19-tagged mCherry luciferase), in vivo effects of the drug combination were studied. These mice express equal basal amounts of Bcl-6 and p53 as GC cell lines. Mice treated with niacinamide 20 mg/kg and romidepsin 2.3mg/kg IP for 5 hours achieved increased acetylation of Bcl-6 and p53, and accumulation of p21 and BLIMP1 compared with controls. Importantly, mice treated with the combination of niacinamide 40 mg/kg and romidepsin 2.3 mg/kg IP achieved decreased tumor burden as measured by bioluminescence signal intensity compared to mice treated with each drug alone and controls. Presently, we are translating these concepts and observations in a proof-of-principle phase I trial evaluating the safety of vorinostat plus niacinamide in lymphoid malignancies. By targeting the specific pathogenetic features of DLBCL, it may be possible to tailor future treatment platforms for discrete subtypes of DLBCL.
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Validation of a Novel Bioluminescent Mouse Model of Sezary Syndrome for Preclinical Drug Screening
Poster Abstract 2725: Sunday, December 11, 2011, 6:00PM
Authors: Salvia Jain, MD and Owen A. O'Connor, MD, PhD, NYU Cancer Institute
Sezary syndrome (SS) is an aggressive leukemic form of cutaneous T cell lymphoma (CTCL) and is generally considered incurable. Until now no true animal model for SS exists which could be used for the screening of novel compounds against the disease. We successfully developed a bioluminescent xenograft mouse model for SS to noninvasively monitor tumor cell engraftment and progression and to measure the effects of treatments on tumor burden. This novel bioluminescent xenograft mouse model of SS enables non-invasive, sensitive, quantitative evaluation of disease progression in living animals and evaluation of pharmacologic factors in real time. We are able to detect and monitor lymphoma cell growth before the presentation of clinical manifestations. Further this model recapitulates our understanding of behavior of drugs used in the treatment of lymphomas such as pralatrexate which has a rapid onset of action compared to romidepsin that has a delayed time to onset of activity. This represents the first bioluminescent animal model of human CTCL that is intended to be used to investigate novel treatment platforms in preclinical studies. This preclinical model also compliments the ongoing phase 2 trial of pralatrexate in relapsed or refractory CTCL. Further in vivo studies to evaluate synergy of promising new agents in this novel mouse model of SS have begun and will be reported.
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Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression
Poster Abstract 1831: Saturday, December 10, 2011, 5:30PM
Authors: Michael Mangone, AB and Hearn J. Cho, MD, PhD, NYU Cancer Institute
Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4-7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM.
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Control of RAG Cleavage Activity Contributes to Maintaining Genome Stability During V(D)J Recombination
Poster Abstract 2416: Sunday, December 11, 2011, 6:00PM
Authors: Susannah Hewitt, PhD and Jane Skok, PhD, NYU Cancer Institute
Acute lymphoblastic leukemia (ALL) results from malignancy of lymphoid progenitor cells and affects both adults and children. It is the most common childhood cancer and despite advances in treatment that now result in above 80% cure rates for children, considerable problems remain with current therapies. These include low cure rates in children with high-risk ALL, the complexity and toxic effects of current treatments and the stubbornly poor prognosis of adults with ALL (with a less than 40% long-term survival rate). ALL can be initiated by errors in V(D)J recombination, a process which creates multiple combinations of receptor genes in B and T lymphocytes in order to target foreign pathogens. During recombination, DNA double strand breaks are introduced at the borders of two selected gene segments and repair creates a new gene combination. Chromosomal translocations can occur both by mis-targeting of the RAG recombinase proteins at cryptic recombination signal sequences, as well as illegitimate repair with a DNA break generated by alternative cellular processes. Our work has unveiled a remarkable and previously unknown control step which acts during V(D)J recombination to protect genome stability. We demonstrated that the key DNA damage response factor and serine/threonine kinase ATM (ataxia telangiectasia mutated), prevents aberrant cleavage during V(D)J recombination. In wild-type cells only one of the two homologous Ig alleles is normally cleaved at a time, whereas in ATM deficient cells both Ig alleles can be cleaved simultaneously and chromosomal aberrations are detected on two Ig alleles (Hewitt et al., Nature Immunology 2009). Our recent work has been directed at understanding how ATM and the RAG recombinase (RAG1 and RAG2 proteins) cooperate to implement allelic control of V(D)J recombination. We hypothesized that ATM may act to control RAG cleavage, either directly or indirectly. To test this, we investigated developing B cells from coreRAG1 or coreRAG2 mice; these are the shortest active forms of the proteins but lack regulatory domains. We assessed mono- versus biallelic cleavage using γH2AX to indicate repair foci and as a read-out for DNA double strand breaks. In pre-B cells from coreRAG1 mice, γH2AX foci were predominantly colocalized with only one Igk allele per cell, which indicates monoallelic cleavage. In contrast, biallelic colocalization was highly significant in coreRAG2 expressing pre-B cells. We have analyzed RAG2 mutants to precisely identify the protein motifs that regulate cleavage. These were introduced into Rag2-deficient pre-B cell lines by retroviral infection. Expression of a coreRAG2 construct in these cells recapitulated the biallelic cleavage seen in ex-vivo isolated pre-B cells. We found that mutation of putative serine/threonine phosphorylation motifs also resulted in significant biallelic colocalization of γH2AX with Igk alleles. This suggests that RAG2 performs a similar function to ATM in restricting simultaneous RAG cleavage on the antigen receptor loci and may indeed cooperate with serine/threonine kinases. These data provide a mechanistic basis for the similarities in chromosomal abnormalities between Atm–/– and coreRag2/p53–/– lymphomas and will contribute to our understanding of why recurrent chromosomal translocations and lymphoid cancers arise in ATM-deficient mice and humans.
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