Lymphoma Clinical Trials Research Articles

Ibrutinib-Resistant Mantle Cell Lymphoma Undergoes Metabolic Reprogramming Towards Oxphos

IntroductionMantle cell lymphoma (MCL) is an incurable B-cell lymphoma subtype and constitutive activation of the B-cell receptor pathway is a hallmark of B-cell lymphomas. Bruton's tyrosine kinase (BTK) is a critical component of the B-cell receptor pathway, and ibrutinib, a first-in-class, once-daily, and oral covalent inhibitor of BTK, was developed to reduce/silence B-cell receptor pathway activity, leading to clinically remarkable anti-tumor activity. In our prior multiple-center Phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68% (Wang et al., NEJM, 2013), surpassing the effectiveness of other therapies. Although ibrutinib is extremely efficacious in patients with relapsed/refractory MCL, the one-year overall survival rate of ibrutinib-exposed patients who relapse is only 22%.MethodsPatient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with reverse phase protein array (RPPA) analysis of ibrutinib-sensitive and -resistant MCL cell lines. Metabolic assays including the measurement of mitochondria respiration rates with the Seahorse analyzer and reactive oxygen species (ROS) levels, targeted metabolomics, and ATP analysis. Functional studies targeting this molecular pathway were conducted, including in vitro cell viability and apoptosis assays, as well as in vivo efficacy studies in an ibrutinib-resistant MCL patient-derived xenograft mouse model.ResultsWES data analysis identified frequent inactivating somatic alterations in ATM, KMT2D, and TP53 in both the ibrutinib-sensitive and -resistant tumors. CDKN2A (5/7, 71%) was frequently deleted, and the deletion was only observed in the ibrutinib-resistant tumors (p = 0.010). The RNA-seq analysis identified a total of 63 protein-coding genes as the most differentially expressed genes (DEGs) between the ibrutinib-resistant and -sensitive groups, with a fold change of ≥ 2 or ≤ -2 and the false discovery rate (FDR q-value) ≤ 0.01. Among the DEGs, 26 genes were upregulated in ibrutinib-resistant tumors. In addition, gene set enrichment analysis (GSEA) revealed the marked upregulation of oncogenic pathways including c-MYC, mTOR (mTORC1), Wnt, and NF-ĸb signaling, followed by cell cycle, apoptosis, BCR signaling and DNA repair in the ibrutinib-resistant tumors. Notably, in addition to these oncogenic pathways, the metabolic pathways, including oxidative phosphorylation (OXPHOS), were significantly enriched in the ibrutinib-resistant tumors (normalized enrichment score > 3 and FDR q-value < 1e-5). In further support of this finding, metabolomics analysis and the measurement of ATP production and mitochondrial respiration indicated that the OXPHOS pathway is the predominant metabolic pathway employed by ibrutinib-resistant MCL cells. To determine the effects of targeting these pathways, OXPHOS was inhibited with a novel electron transport complex I inhibitor (IACS-010759, developed by MD Anderson Cancer Center) in both MCL cell lines and ibrutinib-resistant MCL patient-derived xenograft (PDX) models. Single agent IACS-010759 treatment at 10 mg/kg oral gavage for 5 consecutive days/week completely prevented tumor growth compared with the vehicle control as shown by measuring tumor volume (n = 5, p < 0.0001) and human β2M levels (n = 5; p < 0.0001) throughout treatment. No apparent toxicities were observed in the IACS-010759-treated MCL PDX mice.ConclusionThis current study warrants the exploitation of active cancer metabolic pathways, especially OXPHOS, to improve the clinical outcomes of MCL and additional lymphoma, which is actively being investigated in a Phase I lymphoma clinical trial (NCT03291938). DisclosuresWang:AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria.

In Situ Vaccination with Cpg and Anti-OX40 Antibody: Preclinical Optimization for Clinical Translation

In-situ vaccination is a local intervention in which immune enhancing agents are injected locally into one site of tumor, triggering a T cell immune response locally that then travels to attack cancer throughout the body.We have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting systemic immune response, if any, is detected by the regression of the distant, untreated tumor.In this test for abscopal therapeutic effects, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG) - a TLR9 ligand - and agonist anti-OX40 antibody has provided impressive results. This combination lead to durable disease control and long-term treatment-free survival in multiple mouse models of cancer. CpG induced myeloid cells to secrete cytokines, which subsequently induced OX40 expression on T cells.Thus, we hypothesized that administration sequence and timing may affect the anti-tumor responses of in-situ vaccination.In order to screen for the best sequence and timing we implanted A20 lymphoma tumors bilaterally in opposite sides of the abdomen of Balb/C mice. After tumors were established, one tumor was injected at the selected sequence and timing with the test agents and the resulting immune response was monitored by the measuring growth of the distant, untreated tumor.As opposed to our usual schedule of three injections, even a single injection of CpG (50µg) and anti-OX40 (8µg) resulted in a fully protective systemic immune response. In addition, the cured animals were protected from re-challenge with the same A20 tumor but not unrelated tumors.Decreasing the dose even further to 10µg CpG and 1µg anti-OX40 partially preserved the therapeutic response with a long-term survival of 60%. Concurrent administration of CpG and anti-OX40 resulted in eradication of both local and distant disease. Sequential administration of CpG followed by anti-OX40 preserved the therapeutic efficacy. However, the opposite order of anti-OX40 followed by CpG significantly attenuated the therapeutic effect. While CpG followed by a 24- or 48-hour-delayed anti-OX40 treatment preserved the therapeutic efficacy, a 72h delay in anti-OX40 administration resulted in reduced therapeutic effect.These data demonstrate the importance of the administration sequence for fully protective anti-tumor immune responses. Our data suggest that the anti-OX40 antibody should be administered at the same time as CpG or with only a slight delay but not in the reverse order.Low-dose radiotherapy (2×2 Gy) is an effective treatment for patients with indolent non-Hodgkin's lymphoma. This treatment results in high response rates at the treated site. Since immune infiltrating cells in the tumor microenvironment are essential for in situ vaccination of CpG and anti-OX40 we aimed to assess the effect of adding radiation in our pre-clinical models of lymphoma. We found that the addition of 2x2 Gy radiation did not interfere with the induction of a protective immune response by of CpG and anti-OX40.Given the effectiveness of low dose radiotherapy for local control and its lack of interference with the immune related abscopal response in the pre-clinical model, we are including radiation in our current clinical trials. In addition, we have incorporated our findings in the preclinical model regarding dosing and scheduling of CpG and anti-OX40 antibody to the design of our current in situ vaccination lymphoma clinical trial. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rapid, real time pathology review for ECOG/ACRIN 1412: a novel and successful paradigm for future lymphoma clinical trials in the precision medicine era

ECOG/ACRIN 1412 (E1412) is a randomized, phase II open-label study of lenalidomide/RCHOP vs. RCHOP alone in adults with newly diagnosed de novo diffuse large B-cell lymphoma (DLBCL) and requires NanoString gene expression profiling (GEP) for cell-of-origin testing. Because of high ineligibility rate on retrospective expert central pathology review (ECPR), real-time (RT) ECPR was instituted to confirm diagnosis and ensure adequate tissue for GEP prior to study enrollment. Goal was notification of eligibility within 2 working days (WD). Initially, 208 patients were enrolled, 74 (35.6%) of whom were deemed ineligible by retrospective ECPR. After initiation of RT-ECPR, 219 patients were registered. Of these, 73 (33.3%) were ineligible and were declined enrollment; 47 (21.5% of total) had an ineligible diagnosis on RT-ECPR, and 26 (11.9% of total) had inadequate tissue. Because the 73 ineligible patients were never enrolled, no study slots were “lost” during this phase. Notification of eligibility occurred in an average of 1 WD (Range 0–4) with 97.3% within 2 WD. This novel RT-ECPR serves as a model for future lymphoma trials. Real-time ECPR can help to reduce costs and ensure that study slots accurately reflect the targeted population. In the precision-medicine era, rapid collection of relevant pathology/biomarker data is essential to trial success.

Minimal Residual Disease Assessment in Lymphoma: Methods and Applications.

Standard methods for disease response assessment in patients with lymphoma, including positron emission tomography and computed tomography scans, are imperfect. In other hematologic malignancies, particularly leukemias, the ability to detect minimal residual disease (MRD) is increasingly influencing treatment paradigms. However, in many subtypes of lymphoma, the application of MRD assessment techniques, like flow cytometry or polymerase chain reaction-based methods, has been challenging because of the absence of readily detected circulating disease or canonic chromosomal translocations. Newer MRD detection methods that use next-generation sequencing have yielded promising results in a number of lymphoma subtypes, fueling the hope that MRD detection may soon be applicable in clinical practice for most patients with lymphoma. MRD assessment can provide real-time information about tumor burden and response to therapy, noninvasive genomic profiling, and monitoring of clonal dynamics, allowing for many possible applications that could significantly affect the care of patients with lymphoma. Further validation of MRD assessment methods, including the incorporation of MRD assessment into clinical trials in patients with lymphoma, will be critical to determine how best to deploy MRD testing in routine practice and whether MRD assessment can ultimately bring us closer to the goal of personalized lymphoma care. In this review article, we describe the methods available for detecting MRD in patients with lymphoma and their relative advantages and disadvantages. We discuss preliminary results supporting the potential applications for MRD testing in the care of patients with lymphoma and strategies for including MRD assessment in lymphoma clinical trials.

General Biomarker Recommendations for Lymphoma.

Lymphoid malignancies are a heterogeneous group of tumors that have distinctive clinical and biological behaviors. The increasing prevalence of disease reflects both treatment advances and the fact that some of these tumors are indolent. The ability to determine treatment needs at diagnosis remains problematic for some of the tumors, such as in follicular lymphomas. Major clinical advances will likely depend on precision oncology that will enable identification of specific disease entities, prognostic determination at diagnosis, and identification of precise therapeutic targets and essential pathways. However, refinement in diagnostic evaluation is an evolving science. The ability to determine prognosis at diagnosis is variable, and for many of the lymphoid malignancies prognosis can only be made after initial treatment. Clinical trials that aim to evaluate specific features of these diseases are required in order to advance clinical practice that meaningfully addresses this important public health challenge. Herein, we describe the process and general recommendation from the National Cancer Institute (NCI) clinical trials planning meeting in November 2014 to address clinical trial design and biomarker proposals in the context of NCI-supported lymphoma clinical trials in the National Clinical Trials Network.

CC-122 Repairs T Cell Activation in Chronic Lymphocytic Leukemia That Results in a Concomitant Increase in PD-1:PD-L1 and CTLA-4 Immune Checkpoint Expression at the Immunological Synapse

Immunomodulatory drugs (IMiDs®) such as lenalidomide and immune checkpoint blockade (ICB) antibodies can enhance autologous anti-tumor T cell immunity and have the potential to elicit durable control of disease in B cell malignancies. These immunotherapies are likely to be most effective when employed in treatment combinations. Thus, the goal of pre-clinical research should be to reveal mechanisms of action (MOA) in the tumor microenvironment (TME) and identify biomarkers to guide development of combination therapy for patients. CC-122 is a novel first-in-class pleiotropic pathway modifier (PPM®) that has potent anti-proliferative, anti-angiogenic and immunomodulatory activities and is currently in Phase I clinical trials for lymphoma and chronic lymphocytic leukemia (CLL). Here, we have utilized the immunological synapse bioassay to examine T cell interactions with CLL tumor cells (modeling anti-tumor T cell responses in the TME) following CC-122 treatment and measure the expression of co-signaling complexes at the synapse.Conjugation assays and confocal imaging were used to visualize intercellular conjugate interactions and F-actin polymerization at the immune synapse between CD4+ and CD8+ T cells and autologous CLL tumor cells pulsed with superantigen (acting as antigen-presenting cells, APCs). Peripheral blood was obtained from treatment naive CLL patients (n=40) representative of disease heterogeneity. Treatment of both purified CLL cells and CD4+ or CD8+ T cells with CC-122 (0.01 - 1 μM for 24h) dramatically enhanced the number of T cells recognizing tumor cells (% conjugation) and increased the formation of F-actin immune synapses (area, μm2) compared to vehicle treated cells (P<.01). Notably, CC-122 treatment induced T cells to engage in multiple tumor cell synapse interactions that were more pronounced in restored CD8+ T cell lytic synapses. This immunomodulatory activity was detected across all CLL patient samples and drug concentrations tested. In addition, synapse strength as measured by total fluorescence intensity of F-actin per T cell:APC conjugate increased significantly with CC-122 (P<.01). A critical MOA of lenalidomide is activation of T cell immune synapse signaling. Here, our comparative studies revealed that CC-122 (0.1 - 1 μM) significantly enhanced autologous T cell synapse activity in CLL by 4 - 5 fold versus vehicle (P<.01), whereas lenalidomide (1 μM) enhanced activity by 3 fold vs vehicle. Moreover, CC-122 treatment resulted in increased expression and polarization of tyrosine-phosphorylated proteins at T cell synapses compared to lenalidomide and vehicle treatment (P<.01). This data provides evidence that CC-122 induces functional T cell synapses that control the assembly of signaling complexes between the T cell receptor (TCR) and the F-actin cytoskeletal layer. Following T cell recognition of APCs, co-signaling receptors co-localize at the immune synapse where they synergize with TCR signaling to promote (co-stimulatory receptors) or inhibit (co-inhibitory/'immune checkpoint' receptors) T cell activation. Quantitative image analysis studies revealed that restoration of T cell synapse activity with CC-122 was accompanied by an increased recruitment of inducible co-stimulator (ICOS) to the synapse that was dose-dependent (P<.01). CC-122 treatment also increased polarized expression of CTLA-4 and PD-1 immune checkpoint proteins at the synapse with PD-L1+ tumor cells. The observed up-regulation of co-inhibitory receptors led to combining CC-122 with anti-PD-L1, anti-PD-1 or anti-CTLA-4 blocking antibodies. Results show that these treatment combinations increased T cell synapse activity compared to using these immunotherapies alone (P<.01).In conclusion, our results demonstrate for the first time that CC-122 can activate T cell immune synapse signaling against autologous CLL tumor cells and this immunomodulatory capability is more potent than lenalidomide. We further show that CC-122 activation of T cells is associated with enhanced expression of the co-stimulatory receptor ICOS and co-inhibitory checkpoints CTLA-4 and PD-1 at the synapse site. Importantly, our pre-clinical data demonstrates that this regulatory feedback inhibition can be exploited by the addition of anti-PD-L1, anti-PD-1 or anti-CTLA-4 ICB to CC-122 to more optimally stimulate T cell activity against immunosuppressive tumor cells. DisclosuresHagner:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment. Gandhi:Celgene: Employment, Equity Ownership. Ramsay:MedImmune: Research Funding; Celgene: Research Funding.

Olaparib Recommendations for Ovarian Cancer Patients

Peter Johnson speaks to Gemma Westcott, Commissioning Editor: Peter Johnson is Professor of Medical Oncology at the University of Southampton and Chief Clinician for Cancer Research UK. He graduated from Cambridge University and St Thomas's Medical School (UK). He trained in oncology at St Bartholomew's Hospital, London, where he was an Imperial Cancer Research Fund (ICRF) Clinical Research Fellow and completed his doctoral research on the Bcl-2 gene, its potential as a therapeutic target in lymphoma and the effects of CD40 ligation on the B-cell surface. He was subsequently a Senior Lecturer in Medical Oncology in the ICRF Cancer Medicine Research Unit, Leeds and took up the Chair of Medical Oncology in Southampton (UK) in 1998. He is responsible for bringing together a broad multidisciplinary group of basic, translational and clinical researchers, and linking the research of the academic unit to the extensive clinical practice in cancer treatment in the Southampton Cancer Centre. His research interests are in applied immunology and immunotherapy, lymphoma biology and clinical trials. He is Chief Investigator for lymphoma trials ranging from first in man novel antibody therapeutics to international randomized studies, and for the Cancer Research UK Stratified Medicine Programme. He was Chair of the UK National Cancer Research Institute Lymphoma Group from 2005 to 2011 and has been a member of national trials committees for the Medical Research Council, Cancer Research UK and Leukaemia and Lymphoma Research.

The exclusion of people living with HIV (PLWH) from clinical trials in lymphoma.

Background:The prognosis of non-Hodgkin lymphoma and Hodgkin lymphoma is not affected by HIV serostatus, yet people living with HIV (PLWH) are frequently excluded from clinical trials in lymphoma.Methods:The UK NIHR Clinical Research Network Study Portfolio website was used to identify all the open clinical trials in lymphoma in the United Kingdom in January 2015. Trials that excluded PLWH were further investigated to evaluate if the exclusion was justified by scientific evidence.Results:We identified 56 multicentre open clinical trials in lymphoma including 46 interventional trials. People living with HIV were excluded from 32 interventional trials (70%). We identified a biologically valid reason (a potential increased risk of greater immunosuppression) for excluding PLWH from one trial and possibly for one optional arm in another study.Conclusions:There was no scientific or safety justification for excluding PLWH from most lymphoma clinical trials included on the NIHR portfolio. A clear justification for excluding PLWH was not offered in the available protocols. The exclusion of PLWH should be explicitly justified on scientific grounds in protocols to minimise stigmatisation.

Abstract 1756: Preclinical activity in non-Hodgkin's lymphoma of Selinexor, a selective inhibitor of nuclear export (SINE), is enhanced through combination with standard-of-care therapies

Abstract The nuclear export protein Exportin 1 (XPO1) is overexpressed in diffuse large B-cell lymphoma (DLBCL), follicular small cell cleaved Lymphoma (FSCCL) and a wide variety of other cancers, which often correlates with poor prognosis. Selinexor is an oral SINE currently in Phase 1/2 clinical testing, which targets XPO1 to induce apoptosis across a broad spectrum of tumor types. This broad action is primarily due to forced nuclear retention and reactivation of tumor suppressor proteins (TSPs), resulting in selective tumor cell death. We have previously reported anti-lymphoma activity of selinexor in murine models of DLBCL, in spontaneous canine aggressive lymphomas and in preliminary results from B-cell lymphoma patients enrolled in a Phase 1 trial of hematological malignancies. One promising finding thus far is that efficacy of selinexor appears to be independent of the presence of the mutations in MYC and BCL2/6 found in “double-hit” DLBCL, which is typically resistant to standard treatments. Here we report combination studies involving Selinexor-Dexamethasone and Selinexor-mTOR inhibitor everolimus in Non Hodgkin's lymphoma relative to front-line standard of care. Selinexor was found to be potently cytotoxic across a broad panel of B-cell lymphoma cell lines. Furthermore, combination of selinexor with dexamethasone (Dex) or the mTOR inhibitor everolimus resulted in synergistic cytotoxicity in the WSU-DLCL2 and WSU-FSCCL cell lines. The superior cytotoxicity observed in the combination was consistent with enhanced nuclear retention of different TSPs. In WSU-DLCL2 xenografts, analogs of selinexor KPT-251 and KPT-276 showed comparable efficacy to R-CHOP and in a model of disseminated follicular lymphoma using WSU-FSCCL cells, selinexor had similar efficacy to rituximab for extending survival. The combination of selinexor and Dex or selinexor and everolimus in both xenografts are ongoing. B-cell lymphoma cells in culture are highly sensitive to selinexor. Furthermore, combination of selinexor with Dex or everolimus is synergistically toxic to DLBCL cells. This synergism is consistent with the mechanism of action of these drugs in that both selinexor and Dex antagonize NF-κB and mTOR is an XPO1 cargo protein. The comparable in vivo activity of selinexor and related SINE compounds relative standard of care is consistent with the observations of objective responses in B-cell lymphoma patients in Phase I clinical testing. Ongoing preclinical work is designed to extend comparisons of selinexor with other standard of care and broaden the characterization of combination therapies in an effort to provide a rational basis for studying specific combinations in B-cell lymphoma clinical trials. Citation Format: Asfar S. Azmi, Amro Aboukameel, Robert O. Carlson, Sivan Elloul, Sharon Shacham, Michael Kauffman, Ran Frenkel, Ramzi M. Mohammad. Preclinical activity in non-Hodgkin's lymphoma of Selinexor, a selective inhibitor of nuclear export (SINE), is enhanced through combination with standard-of-care therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2015-1756

Preclinical Activity of a New Proteasome Inhibitor, MLN9708, in Acute Myelogenous Leukemias Expressing NPM1 Mutated Protein

ABSTRACT: The nucleophosmin-1 (NPM1) mutation represents the most common genetic lesion (30-35%) in adult acute myeloid leukemia (AML) with the mutation resulting in the cytoplasmic delocalization of the NPM protein (NPMc+, most commonly mutation type A, c.860_863dupTCTG). Outside of conventional chemotherapy, treatment options for those with relapsed disease are extremely limited. We have previously discovered that leukemic cells expressing NPMc+ are preferentially sensitive to the cytotoxic effects of the first generation proteasome inhibitor, bortezomib, as result of the induction of superoxide in these cells (Huang M, et al., Leukemia, 2013). However, bortezomib has demonstrated only modest anti-leukemic activity as a single agent in early phase clinical trials that have included primarily heavily pretreated patients not stratified according to mutational genotype. In addition, the high incidence of clinically significant peripheral neuropathy in these studies led to early discontinuation of treatment. MLN9708 (Ixazomib) is a novel second generation proteasome inhibitor that is orally bioavailable and has shown promising activity and improved tolerability in early phase clinical trials in lymphoma and in multiple myeloma, where it is currently in a Phase III clinical trial. The efficacy of MLN9708 in relapsed patients with AML has not yet been examined. We have investigated the in vitro effects of MLN9708 on the cytotoxicity, generation of reactive oxygen species (ROS), and cellular glutathione in both cultured NPMc+ AML cell lines and primary leukemic samples. Cultured OCI-AML3 cells (human AML cell line featuring the NPMc+ mutation) were approximately 50-fold more sensitive to MLN9708 with a mean half inhibitory concentrations (IC50 ± SD) of 67 nM ± 1.83 nM at 24 hours than were an AML cell line (THP-1) expressing wild type NPM1. Knockdown of both NPM1/NPMc+ by inducible NPM1 shRNA expression significantly attenuated the MLN9708-mediated cytotoxicity, as determined by MTS colorimetric and flow cytometric apoptosis assays. As with bortezomib, MLN9708 treatment at 75 nM induced a 2.1-fold (p < 0.002) increase in superoxide-specific DHE (PE)-derived fluorescence as compared to controls. Pre-incubation of cells with 25 mM N-acetylcysteine, a ROS scavenger, dramatically reduced cytotoxicity, strongly implicating the generation of ROS in the mechanism of MLN9708-induced cytotoxicity. Knockdown of NPM1/ NPMc+ expression resulted in an increase of glutathione levels of 2.6-fold (p < 0.04) in untreated cells, and 2.3-fold (p < 0.005) and 2.9-fold (p < 0.004) after a 22 hour incubation with MLN9708 at pharmacologically achievable doses of 75 nM and 150 nM, respectively (Figure 1). In addition, a concomitant 4-fold reduction (p < 0.04) in the NADP+/NADPH ratio was observed in the NPM1/NPMc+ depleted OCI-AML3 cells. In vitro apoptosis assays of primary leukemic blasts isolated from five patients with relapsed NPMc+ AML showed an IC50 for MLN9708 ranging from 49.963-467.487nM (183.8 nM ± 176.8 nM). Based on these promising in vitro studies, a Phase 2 clinical trial of single-agent oral MLN9708 (clinicaltrials.gov identifier: NCT02030405) has been initiated for patients with relapsed or refractory NPMc+ AML.We conclude that the expression of NPMc+ renders AML cells more susceptible to MLN9708 toxicity and that this effect results at least in part from the generation of superoxide. Lower glutathione levels in NPMc+ expressing cells suggests an underlying defect in the antioxidant defense pathway. Demonstration of the sensitivity of NPMc+ AML cells to MLN9708 as a single agent may serve as a platform for the development of novel combination strategies with this drug.Acknowledgments: Research was facilitated by the ASH Clinical Research Training Institute and AACR/ASCO Clinical Cancer Research training program. J.S.G is funded by the Leukemia & Lymphoma Society Career Development Award and ASCO YIA award through a grant supported by Mr. Aaron Sasson. J.S.G. is further supported by the Stanford Hematology Fellowship Program. [Display omitted] DisclosuresOff Label Use: MLN9708 is an investigational compound by Millennium Pharmaceuticals, Inc. (MPI). Through an MTA we have secured the compound for in vitro analysis. This data has led to a clinical trial and drug for that study is supplied by MPI.. Medeiros:Agios: Consulting - Ad board Other.

HDAC Inhibitors As Potential New Agents Improving the Efficacy of Monoclonal Antibodies

HDAC (histone deacetylase) inhibitors belong to a novel class of antitumor agents widely explored in hematology. So far two of them - romidepsin and vorinostat have been registered in cutaneous T-cell lymphoma, while panobinostat awaits FDA decision concerning its registration in multiple myeloma. Several other agents are currently being tested in clinical trials in lymphoma and myeloma patients and preliminary results suggest that they may be most effective when used in combinations with other anti-cancer drugs. Reports from others (Sugimoto et al. Biochem Biophys Res Commun 2009 Shimizu et al. Leukemia 2010) and the results from our group show that HDAC inhibitors augment the efficacy of anti-CD20 monoclonal antibodies (mAbs) in complement-dependent cytotoxicity (CDC) by up-regulating CD20 level. In our previous reports we have shown that HDAC6 isoform is involved in the regulation of CD20 degradation and trafficking. Since the efficacy of mAbs depends not only on the level of target molecule but is also regulated by the expression of complement regulatory proteins (mCRPs) - CD46, CD55 and CD59, we sought to investigate the influence of HDAC inhibitors on the expression of those molecules. Moreover, given the fact that HDAC inhibitors have been reported to regulate NK cells activity, we investigated their influence on antibody-dependent cell-mediated cytotoxicity (ADCC) which is one of the mechanisms of action of mAbs in vivo.The purpose of this study was to thoroughly investigate the influence of HDAC inhibition on the level of surface molecules – CD20 and CD38 – targets already used and tested in clinical trials in immunotherapy of B-cell malignancies as well as the expression of mCRPs. Using non-selective pan-inhibitors as well as selective inhibitors of particular HDAC classes and isoforms we sought to show the inhibition of which HDAC enzyme is the most effective in potentiating the outcome of mAbs based anti-tumor therapies. Since the results of clinical trials show that the use of HDAC inhibitors has several non-specific adverse effects, we hypothesize that selective inhibition of particular isoforms may considerably diminish these toxicities.Using flow cytometry we performed experiments assessing the level of surface CD20 CD38 and mCRPs – CD46, CD55 and CD59. We used established lymphoma, chronic lymphocytic leukemia (CLL) and myeloma cell lines as well as primary cells isolated from the patients suffering from CLL. We observed that the use of HDAC pan-inhibitors – trichostatin A (TsA) and vorinostat (Fig.1A) and selective HDAC6 inhibitors – tubacin and ACY1215 (ricolinostat) (Fig.1B) considerably improves the outcome of anti-CD20 mAbs – rituximab and ofatumumab in CDC in several B-cell lymphoma cell lines and in CLL primary samples (Fig. 1C). In our experiments we show that selective inhibition of HDAC6 in Raji cells does not influence the expression of mCRPs (Fig. 1D). However, we observed that pre-incubation with tubacin considerably increases the expression of CD38 (Fig. 1 D). Therefore, in our further studies we aim at expanding our observations to primary samples from multiple myeloma patients, especially since novel anti-CD38 mAbs are currently intensively explored in the therapy of this malignancy.Moreover, using flow cytometry we determined the influence of HDAC inhibition on degranulation of NK cells isolated from healthy donors in ADCC in vitro assay with rituximab and ofatumumab. Contrary to the reports of others, we observed no negative regulation of NK cells activity and their ability to kill target tumor cells. Therefore, since HDAC inhibitors seem to influence mainly CDC, we investigated the influence of specific HDAC inhibitors on the level of mCRPs. Those results suggest that specific inhibition of HDAC3 (Fig. 1E) and HDAC1/2 (Fig. 1F) are effective therapeutic strategies as they considerably decreased the expression of complement inhibitors and concomitantly increased the level of CD20 antigen. However, further studies including silencing of particular HDAC isoforms with shRNA are needed to confirm these findings. Altogether, our results strongly suggest that HDAC inhibitors potentiate the outcome of mAbs used in hematological malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Simple Scoring System for Identifying Relapsed/Refractory Lymphoma Patients Prematurely Withdrawn from Phase I Trials: The Gustave Roussy Experience

Abstract Background: Treating relapsed/refractory non-Hodgkin and Hodgkin lymphoma in fit young and elderly patients represents a considerable challenge for clinicians. Combined cytotoxic agents are rapidly ineffective, especially when relapse occurs after autologous stem cell transplantation (ASCT). Alternative regimens are often sparse. In the era of personalized medicine, phase I clinical trials offer an alternative therapeutic option through a multitude of immunotherapy and targeted drugs in development. Phase I trials aim to determine the recommended phase II dose (RP2D) through toxicity and pharmacokinetic assessments. Documenting signal of activity is also a major objective, underlying the importance of including patients susceptible to remain on study until first lymphoma response evaluation (usually 6 weeks, 2 cycles). We aimed to assess a simple scoring system that could identify patients who will discontinue phase I studies before 6 weeks. Patients and Methods: Data from all lymphoma patients treated within a phase I trial in Gustave Roussy (GR) Cancer Center were retrospectively collected. 83 consecutive patients were enrolled in 17 phase I trials at GR between January 2008 and May 2014. All patients had progressive lymphoma at time of enrollment, after a median of 3 prior therapeutic lines (range 1;13). 37 patients (45%) received an ASCT prior to phase I inclusion. Median age was 67 years (range: 23-92) and WHO performance status (PS) was 0 in 36 patients (43%), 1 in 43 patients (52%) and 2 in 4 patients (5%). Median time from lymphoma diagnosis to phase I inclusion was 47 months. Lymphoma histological subtypes were represented as follows: 21% Hodgkin lymphoma, 36% aggressive non-Hodgkin lymphoma (83% diffuse large B cell lymphoma, 17% T cell lymphoma), 24% indolent non-Hodgkin lymphoma (70% follicular lymphoma, 25% marginal zone lymphoma, 5% Waldenström macroglobulinemia) and 19% mantle cell lymphoma. Univariate analysis on this cohort allowed identifying simple factors significantly associated with overall survival (OS). A simple scoring system, predictive for OS was pre-established and validated through a multivariate analysis in 2 large cohorts of various hematological malignancies by our group. Statistical tests were conducted on this relapsed/refractory lymphoma cohort, to evaluate this score's OS and early study discontinuation predictive ability. Results: Within a median follow up of 19 months, median OS and progression free survival (PFS) were respectively 18 (CI95%: 12; 37) and 3 (CI95%: 1.7; 3.6) months. Best overall response rate (ORR) and disease control rate (DCR: objective response and stable disease rates) were respectively 18% and 61%. Thirty-four patients (41%) experienced grade 3 or 4 adverse events and 7 patients (8%) a dose limiting toxicity (DLT). WHO performance status (PS) &gt; 0 at inclusion, baseline albumin ≤ 35 g/l and baseline LDH ≥ 250 UI/L were significantly associated with poorer OS. The predefined GR prognostic score combined 2 simple variables, PS and baseline serum albumin (+1 if PS 0, +1 if albumin≤ 35 g/l). In our lymphoma cohort, patients with a GR score = 0 experienced significantly better OS compared to patients with a score = 1 and a score = 2 (37 months vs 17 months vs 9 months; p = 0.007). This simple score, distinguishes patients most likely to remain on study for more than 6 weeks, considered as the minimum required period for response and toxicity evaluation (classical DLT and targeted therapies associated long term toxicities). Among patients prematurely withdrawn from clinical trials, 91% had a GR score ≥ 1 (p=0,007). Main study discontinuation reason was progressive disease (77%), drug related toxicity was only responsible for study discontinuation in 13 % of cases. Conclusion: Our data demonstrate efficacy and safety of phase I clinical trials in lymphoma, thus offering an interesting alternative therapeutic option for fit young and elderly relapsed/refractory lymphoma patients. The GR simple score, can both help in selecting patients most likely to benefit from a phase I trial (better OS) and to determine the phase II recommended dose (reduced early trial discontinuation). Disclosures No relevant conflicts of interest to declare.

Ibrutinib and Rituximab Are an Efficacious and Safe Combination in Relapsed Mantle Cell Lymphoma: Preliminary Results from a Phase II Clinical Trial

▪Single-agent ibrutinib has been approved by the FDA for patients with mantle cell lymphoma (MCL) who received at least one prior therapy based on a phase II clinical trial in which ibrutinib elicited a response rate of 68% (Wang et al, NEJM, 2013). In this clinical study we found a transient increase in circulating MCL lymphocytes during the initial phase of tumor reduction. We hypothesized that targeting the circulating MCL cells with intravenous rituximab will further improve the efficacy of ibrutinib. We conducted a single-center phase II clinical trial with ibrutinib in combination with rituximab for relapsed MCL with no upper limit for prior lines of therapy. Among 50 patients with MCL, 100% received prior rituximab, 77% received prior Hyper-CVAD, 75% received prior bortezomib, and 20% received prior lenalidomide. Rituximab was dosed at 375 mg/m2 iv weekly X 4 during cycle 1 (cycle = 28 days), then on day 1 of every cycle from 3-8, and thereafter once every other cycle up to 2 years. Ibrutinib was dosed at 560 mg orally daily continuously. With a median follow up time of 6.5 months (range 1-10), 45 patients are evaluable for toxicity and efficacy as of July 21, 2014. Thirty three patients (73% of evaluable patients) have Ki-67 < 50%. Seventeen (17) patients are now off study including 2 patients with secondary malignancies (AML and lung cancer). One (1) patient in CR withdrew consent due to social issues and continued on commercial ibrutinib. Two (2) patients in remission withdrew consent due to their concerns that rituximab-ibrutinib might worsen their atrial fibrillation and both continued on single-agent commercial ibrutinib. One patient was off study due to bleeding. Three (3) patients in remission went off to stem cell transplantation. Eight (8) patients are off study due to progressive MCL (4 never responded: 4 responded then progressed), all of them had Ki-67 greater than 50% (range 50-100%). There were no toxic deaths due to therapy. Grade 3 hematologic toxicity events included neutropenia (1) and thrombocytopenia (1). The most common (≥ 20%) grade 1-2 non-hematologic toxicity events regardless of its relationship with study therapy included fatigue (18), diarrhea (11), myalgia (11), dyspnea (11), blurred vision (10), nausea (9),dry eye (9) and atrial filbrillation (6). The efficacy data is listed in Table 1. The ORR to date is 87% with CR in 17 patients (38%) and PR in 22 patients (49%). The CR rate is high in this study in the context of historical data (21% by single-agent ibrutinib). Median duration of response and PFS has not been reached. Notably, all 10 patients with SD (2) and PD (8) have Ki-67’s ≥ 50%. Excluding the 12 out of 45 evaluable patients with Ki-67 ≥ 50%, the ORR for 33 patients with lower Ki-67 (< 50%) is 100% (48% for CR and 52% for PR) in patients with relapsed/refractory MCL. While this trial is ongoing, preliminary data indicated that Ibrutinib-rituximab combination is well-tolerated and is efficacious, especially in patients with Ki-67 less than 50%.Table 1The best response related to Ki-67All n (%)Ki-67 < 50%Ki-67 ≥ 50%Evaluable patients453312ORR39 (87%)33 (100%)6 (50%)CR17 (38%)16 (48%)1 (8%)PR22 (49%)17 (52%)5 (42%)SD2 (4%)02 (17%)PD4 (9%)04 (33%)Duration of responseNRNRNRPFSNRNRNR DisclosuresWang:Pharmacyclics and Janssen: Honoraria, Research Funding. Off Label Use: Ibrutinib and Rituximab for mantle cell lymphoma clinical trial. Westin:Pharmaciclics and Janssen: Honoraria, Research Funding. Fayad:Pharmacyclics and Janssen: Research Funding. Samaniego:Pharmacyclics and Janssen: Research Funding. Romaguera:Pharmacyclics and Janssen: Research Funding.

Abstract A04: Measuring autophagy inhibition as a pharmacodynamic response to hydroxychloroquine in a spontaneous canine lymphoma clinical trial

Abstract Background: (Macro) Autophagy is the bulk, lysosomal degradation process of organelles and long-lived cytosolic proteins via sequestration into double membraned vesicles called autophagosomes. In recent years, autophagy inhibition has become an attractive, anti-cancer therapy as numerous studies have provided evidence for autophagy as a mechanism of survival and resistance. The most widely used autophagy inhibitors in clinical trials are chloroquine (CQ) or hydroxychloroquine (HCQ), due to their well-established safety profiles after decades long use as anti-malarial therapeutics. However, since measuring autophagy modulation is difficult in vivo, there is still uncertainty as to whether autophagy inhibition by CQ or HCQ is achievable in tumors. In order to determine the efficacy of HCQ in combination with doxorubicin (DOX) and to obtain measures of pharmacodynamic (PD) response, a clinical trial for canine patients with lymphoma was undertaken. Dogs present a unique model for studying human cancer as they inhabit the same environment and their cancers also arise spontaneously. In addition, canine clinical trials progress more rapidly than human trials, and samples can be collected more readily in dogs. Material and Methods: After an initial phase I dose escalation study, twelve canine patients presenting with lymphoma were enrolled into the study. Patients received 12.5 mg/kg daily HCQ, orally, and received their first round of 25 mg/m2 DOX treatment 3 days later. This dose is a 20% reduction from the standard 30 mg/m2, as dose-limiting neutropenia was observed at the full DOX dose in the dose escalation study. DOX was delivered IV every 21 days, for five treatments. Whole blood, serum, plasma, lymph node aspirates, and two punch biopsies, were collected prior to HCQ administration and 3 days after HCQ, but before DOX delivery. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and were immediately processed for flow cytometry, Western blot (WB), or electron microscopy (EM) analysis. Lymph node aspirates and PBMCs were stained with an LC3 antibody, a marker for autophagosomes, and analyzed by flow cytometry. Western analysis for LC3 and p62, a selective substrate for autophagy degradation, was performed on PBMCs and punch biopsies. Mass spectrometry analysis was used to determine HCQ levels in the plasma and remaining biopsy. Results: HCQ was detectable in both plasma and tumor after administration, with tumor levels about 300 times higher than that of plasma. However, HCQ levels varied widely and no correlation was observed between plasma and tumor levels. Patients with at least a 1.5 fold increase in LC3 I or II, as determined by WB, had significantly higher tumor HCQ levels (p=0.028). Increases in LC3 expression, indicative of inhibited autophagy, also correlated to increased p62 expression (p=0.034 Pearson r = 0.745). Yet, there was no correlation of LC3 expression in PBMCs compared with tumor, nor was there a correlation between plasma HCQ levels and LC3 expression. Additionally, there was no correlation between LC3 expression in PBMCs, as determined by flow cytometry, to expression determined by WB. Autophagosomes were visible by EM in the PBMCs after HCQ treatment. Conclusions: Autophagy inhibition by HCQ was only achieved in those patients with the highest tumor HCQ levels. Flow cytometry does not appear to accurately reflect PD response and it is recommended to use WB or EM analysis instead. Lastly, plasma does not appear to be a good surrogate for tumor PK/PD assessment, and thus, should not be used in lieu of tumor assessment. Citation Format: Rebecca A. Barnard, Luke A. Wittenburg, Daniel L. Gustafson, Andrew Thorburn, Douglas H. Thamm. Measuring autophagy inhibition as a pharmacodynamic response to hydroxychloroquine in a spontaneous canine lymphoma clinical trial. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A04.

Mechanisms of histone deacetylase inhibitor in treating T-cell lymphoma and related clinical trials

Mechanisms of histone deacetylase inhibitor in treating T-cell lymphoma and related clinical trials

Targeting PI3-kinase (PI3K), AKT and mTOR axis in lymphoma.

Targeted therapy represents a transformation in oncology, a field that has relied primarily on non-selective cytotoxic therapies. Phosphatidylinositol 3-kinase (PI3K) is a family of ubiquitous signalling molecules involved in a wide variety of cellular processes and likewise, in a broad selection of human cancers. The discovery that the p110-δ form of PI3K is differentially expressed in normal and malignant lymphocytes has led to the development of specific inhibitors that are currently in clinical trials for lymphoma. Downstream effectors of PI3K, including v-akt murine thymoma viral oncogene homolog 1 (AKT; also termed AKT1) and mechanistic target of rapamycin (serine/threonine kinase) (mTOR) are similarly important in lymphoma, and agents targeting these components of the PI3K-AKT-mTOR axis are also underway, although at earlier stages of development. In this review we examine the role of PI3K-AKT-mTOR in normal and malignant lymphocytes, as well as the preclinical and clinical status of a number of inhibitors of this pathway.

Accrual Of Patients With Relapsed and Refractory DLBCL Onto Clinical Trials

BackgroundNew drug development in diffuse large B cell lymphoma (DLBCL) has become a very active area of research in the last several years with the advent of monoclonal antibodies and targeted therapies. We sought to determine the rate of accrual of patients with relapsed/refractory (R/R) diffuse large B cell lymphoma (DLBCL) onto early phase clinical trials, and hurdles to their enrolment by performing a retrospective analysis of all DLBCL cases diagnosed at our institution from 01/2006 to 03/2012. This time span represents an active time period for clinical trials in lymphoma at our institution. MethodsDLBCL cases were identified through the hospital tumor registry. Patients were included in the analysis if they had any diagnosis ofDLBCL relapsed or refractory to standard therapy. Baseline demographics and disease characteristics, details of treatment, responses, relapse, evaluation for clinical trials and participation in clinical trials were determined by review of hospital charts. Only Phase I and II clinical trials were considered for this analysis. ResultsOf a total of 284 patients, 76 had relapsed/refractory disease, 10 of 25 had a successful autologous stem cell transplant (ASCT), and there is insufficient data on 1 patient. Of the remaining 65, 11 (17%) made it to trial. The median age was 65, 34 were male, median number of prior therapies was 2, 74% had at least one comorbidity and 46% had at least 2. Sixty-two percent of patients had de novo DLBCL, 18% transformed and 20% had a composite lymphoma including DLBCL. Reasons for failing to enroll on trial included prohibitive comorbidity (21%), rapid progression (15%), decision for palliation (15%), prior second malignancy (9%), thrombocytopenia (13%), CNS disease (9%), proximity to ASCT (2%), no protocol available(6%), palliative radiation (6%). Patients on trial tended to be younger (58.4 vs. 65.8 years), and to have a lower IPI (mean 3.1 vs 3.4). There was no difference in the number of prior of therapies (2.31 vs. 2.26) or number of comorbidities (2.18 vs 2.21). However, out of the 11 who made it to trial, 7 patients had failed ASCT (46.7% of patients who failed ASCT) vs. 4 (10%) who never had a transplant (p=0.005, Fisher's exact test). Among the relapsed and refractory cases, 81% of cases were discussed at tumor board. ConclusionsAlthough our study is small, we demonstrate that in an active research center, where a large number of R/R DLBCL patients are discussed at tumor board during which information about clinical trials is disseminated, a minority of patients with DLBCL not responding to standard therapy make it to trial.Patients having failed an ASCT are more successfully enrolled onto clinical trials. Interestingly, a similar accrual rate was seen in relapsed non-small cell lung cancer (Baggstrom,J Thor Oncol 2011) and similar barriers to enrolment were found for solid tumor patients (Lara, JCO 2001). Accrual of R/R DLBCL patients onto clinical trials is possible but challenging. A multi-institution analysis is underway. Disclosures:No relevant conflicts of interest to declare.

Abstract 2215: Pharmacogenomic investigation of Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765): drug sensitivity in diffuse large B-cell lymphoma (DLBCL) within a tumor microenvironment-aligned high-throughput screen.

Abstract Ibrutinib (PCI-32765) is an orally administered small molecule that covalently binds to Cys-481 of Bruton's tyrosine kinase (BTK). Ibrutinib has demonstrated promise across several types of B-cell malignancies and is currently in Phase 2/3 clinical testing. Preclinical studies have established three key mechanisms following blunting of proximal B cell receptor (BCR) signaling through BTK inhibition: (1) suppression of pro-survival pathways, (2) diminished integrin activation and (3) attenuation of chemotactic response. Single agent ibrutinib Phase 1/2 clinical trials have revealed high clinical response rates in both naïve and relapsed or refractory chronic lymphocytic leukemia (CLL), as well as, relapsed or refractory mantle cell lymphoma (MCL) patients (Byrd, et al. 2012 ASH; Wang, et al. 2012 ASH; Advani, et al. 2010 JCO). Response rates in a phase 2 diffuse large B cell lymphoma (DLBCL) clinical trial appeared to be more prevalent in “activated B-cell” (ABC) over “germinal center B-cell like” (GCB) DLBCL patients (Staudt et al., ASH 2012). To gain a further understanding of ibrutinib response and inform optimal therapeutic combinations in DLBCL, we established a combination high throughput pharmacology screen (cHTS) with ibrutinib. Ibrutinib was evaluated alone and in combination with 99 targeted compounds, across 17 (12 GCB; 5 ABC) DLBCL cell lines. To better align with human biology, DLBCL cell lines were screened in the presence of human marrow stromal cell conditioned media (hMSC-CM) and B-cell receptor stimulation via anti-IgG/anti-IgM antibodies. Interestingly, 8/17 (47%) DLBCL cell lines were intolerant to any external BCR stimulation, so those lines were screened in hMSC-CM without exogenous BCR stimulation. Under our experimental conditions, 11/17 (65%) cell lines displayed some sensitivity (IC50 &amp;lt; 10 M) to ibrutinib at 72 hours based on ATPliteTM measurements [breakdown: 4/17 (24%) highly sensitive (IC50 &amp;lt; 200 nM), 2/17 (12%) sensitive (IC50 200 nM &amp;lt; x &amp;lt; 1 M), 5/17 (29%) modestly sensitive (IC50 1 M &amp;lt; x &amp;lt; 10 M), and 6/17(35%) resistant (IC50 &amp;gt; 10 M)]. Consistent with DLBCL clinical responses, the highly sensitive (IC50 &amp;lt; 200 nM) DLBCL models, which better reflect clinically attainable drug concentrations, demonstrated approximately a two-fold enrichment in ABC (40%; 2/5) versus GCB (17%; 2/12) response with single agent ibrutinib. However, selective drug combinations were found to drive greater sensitivity across both ABC and GCB subtypes. Ongoing work is focused on mapping pathways that contribute to combination drug sensitivity by comparing pharmacologic synergy of targeted agents against mutational (Exome-Seq) and whole genome gene expression profiling (GEP). Together, these data will help to inform optimal ibrutinib combination strategies in DLBCL. Citation Format: Cuc Davis, Tineke Casneuf, Willem Lightenberg, Richard Rickles, Winnie Tam, Matthias Versele, Steven McClue, Sriram Balasubramanian, Joseph Buggy, Kate Sasser, Brett Hall. Pharmacogenomic investigation of Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765): drug sensitivity in diffuse large B-cell lymphoma (DLBCL) within a tumor microenvironment-aligned high-throughput screen. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2215. doi:10.1158/1538-7445.AM2013-2215

Abstract 2453: Optimizing a modular synthetic approach for producing SH7139, a new drug candidate for treating B-cell lymphomas.

Abstract The scale-up of synthetic methods for producing small molecule therapeutics can often prove to be problematic and expensive. Synthetic mechanisms can be inefficient and purification methods used to select specific isomers or remove reactants or other contaminants can often be difficult to scale. The modular approach we developed for creating small molecule targeting agents called SHALs (Selective High Affinity Ligands) as therapeutics and diagnostics for B-cell lymphoma circumvents many of these problems by using off the shelf components as “recognition elements” that collectively contribute to the molecules high affinity and selectivity and miniPEG and common amino acids to build up the scaffold using solid phase chemistry. Here we present the results of our efforts to scale up the synthesis of the first of these promising new drug candidates, SH7139, to provide the compounds needed for pre-IND animal testing and a pet dog lymphoma clinical trial. Working with AmbioPharm Inc,, a CRO with extensive expertise in the GMP synthesis of peptide pharmaceuticals, we recently completed small pilot production scale syntheses of SH7139 and several closely related analogs. The simple solid phase peptide-based chemistry used to synthesize the compounds coupled with preparative HPLC adapted well to scale-up and resulted in the production of highly purified compounds identical in structure (as determined by mass spectrometry and nuclear magnetic resonance spectroscopy) to the bench scale syntheses we performed in house. However, one unexpected and notable difference between the scaled-up synthetic products and their bench-scale synthesis counterparts was their solubility. SH7139 and two of its analogs, which are essentially insoluble in water and PBS when synthesized in house, were observed to be highly soluble (&amp;gt;100mg/ml) when synthesized using the scale-up protocol. While the reason for this is still under investigation, microscopic analyses of the dried product suggest the solubility difference may relate to the lyophilization step used to produce the dried product. The majority of the insoluble SH7139 we produce in bench-scale syntheses is amorphous in form, while the soluble SH7139 produced by AmbioPharm appears to be in a semi-crystalline or glass-like state. This research was supported, in part, by NIH/NCI SBIR grant 1R43CA159843-01 to Rod Balhorn. Citation Format: Monique Balhorn, Saphon Hok, Rod Balhorn. Optimizing a modular synthetic approach for producing SH7139, a new drug candidate for treating B-cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2453. doi:10.1158/1538-7445.AM2013-2453

Activity and complexes of mTOR in diffuse large B-cell lymphomas—a tissue microarray study

Diffuse large B-cell lymphoma is a heterogeneous group of diseases with different responses to therapy. Targeting mTOR (mammalian target of rapamycin) offers a new approach to improve the treatment. mTOR inhibitors are being developed and are in clinical use in mantle cell lymphoma therapy and clinical trials are ongoing in other high-grade lymphomas as well. However, there is limited data about mTOR activity and the expression of its different complexes in diffuse large B-cell lymphomas. Tissue microarray blocks were constructed from paraffin-embedded biopsy specimens. More than 700 immunohistochemical stainings (mTOR signaling-related proteins and phosphoproteins, markers for lymphoma classification) were evaluated from 68 diffuse large B-cell lymphoma biopsies from conventionally treated and followed patients. Approximately 30% of cases were characterized as germinal center-derived diffuse large B-cell lymphomas, which showed virtually no mTOR activity, as determined by phospho-ribosomal S6 expression, the most sensitive marker of mTOR activity. In about 80% of non-germinal center-derived diffuse large B-cell lymphoma cases, positivity of mTOR-related phosphoproteins was observed, denoting mTOR activity. Moreover, Rictor (a characteristic protein of the mTOR complex2) was overexpressed in 43% of all diffuse large B-cell lymphomas and in 63% of mTOR-active non-germinal center diffuse large B-cell lymphoma samples. Rictor overexpression with mTOR activity indicated significantly worse survival for patients than mTOR inactivity or mTOR activity with low Rictor expression. These results suggest that mTOR activity is characteristic in most non-germinal center-derived diffuse large B-cell lymphomas with potentially variable mTOR-inhibitor sensitivity. Taken together, mTOR inhibitors may be useful in addition to regular therapy in diffuse large B-cell lymphomas, however, patient and inhibitor selection criteria must be carefully considered.