bluuebunny/crossref_metadata_2025 huggingface dataset:
B Cell Lymphomas Are Resistant towards Bone Morphogenetic Protein (BMP) Induced Growth Inhibition.
Authors: Huse Kanutte , Eide Marianne B. , Kersten Christian , Smeland Erlend B. , Myklebust June H.
Published: October 2019

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily, and mediate their effects mainly through the Smad signalling pathway. Whereas TGF-β is well established as one of the most potent negative regulators in hematopoietic cells, the role of BMPs remains more elusive. We have previously shown that BMP-6 inhibits the growth of naïve and memory human B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that resistance towards BMP-induced growth inhibition is a possible mechanism for lymphomagenesis. In the current study, 7 B cell lymphoma cell lines (representing Burkitt lymphoma (BL) and DLBCL) and tumour material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We analyzed the expression of BMP receptors by FACS analysis, and found variable expression of the BMP receptor type I (Alk2, Alk3 and Alk6) and type II (BMP RII, Activin RIIA and RIIB) among the cell lines and in primary lymphoma cells, suggesting variable binding of BMPs. We next investigated the effect of BMP-2, BMP-4, BMP-6 and BMP-7 on proliferation and survival of B lymphoma cell lines, and found 2 of 7 cell lines to be resistant towards BMP-2 and BMP-4 induced growth inhibition. In contrast, 4 of 7 and 7 of 7 cell lines were resistant to BMP-6 and BMP-7 induced growth inhibition, respectively. In Sudhl6 cells that were highly sensitive to BMP-2 and BMP-6 induced apoptosis and inhibition of proliferation, we demonstrated that the cytokines IL-10, CD40 Ligand and BLyS were able to counteract the negative effects induced by BMPs, while IL-2 and IL-4 were not. On the contrary, both BMP-2 and BMP-6 greatly increased anti-IgM activation induced apoptosis. In resistant lymphoma cells, the BMPs were not able to induce detectable levels or induced low levels of phosphorylated SMAD1/5/8 compared to sensitive cell lines. Low or no increase in phosphorylation of SMAD1/5/8 induced by BMPs could only partly be explained by low/ undetectable expression of BMP receptors. Hence, upregulation of inhibitory Smads (Smad6, Smad7) or mutations in receptors or Smads represent other possible mechanisms for resistance to BMPs in lymphomas, and this is currently under investigation. We also investigated if the lymphoma cells produced BMPs themselves and found that 5 of 7 cell lines and 3 of 5 primary lymphomas produced significant amounts of BMP-7. Some lymphoma cells also had detectable levels of BMP-4 and BMP-6. Our findings that lymphoma cells are resistant towards BMP-7 and to some degree BMP-6 induced growth inhibition, whereas they produce these cytokines, suggest that resistance towards BMP induced signalling in B cell lymphomas can contribute to increased tumour growth.
Value of FDG-PET in Prediction of Splenectomy Findings in Patients with Suspected or Known Lymphoma.
Authors: Rutherford Sarah C. , Andemariam Biree , Philips Shibu M. , Chadburn Amy , Furman Richard R. , Coleman Morton , Goldsmith Stanley J. , Leonard John P.
Published: October 2019

Splenectomy is frequently performed for diagnostic purposes in patients with suspected or known lymphoma, including assessment for possible transformation. The ability to predict splenectomy findings (and potentially avoid associated morbidity and mortality) would be valuable. The purpose of this study was to determine whether preoperative18F-fluorodeoxyglucose positron emission tomography (FDG-PET) results correlate with pathologic diagnosis from splenectomy.

Methods: One hundred sixty-five patients who had undergone a splenectomy at New York Presbyterian Hospital from January 2004 to July 2006 were identified from the pathology database. Records of these patients were searched and 11 with suspected or known lymphoma as an indication for splenectomy and who had a pre-splenectomy PET scan were identified. A nuclear medicine physician performed a blinded review and assigned each PET scan to one of the following categories based on splenic FDG standardized uptake values (SUV): low splenic metabolic activity (correlated with maximum SUV range 2-3.7), intermediate splenic metabolic activity (maximum SUV range 6–7), and high splenic metabolic activity (maximum SUV range 26–29). Findings were correlated with splenectomy pathologic diagnosis.

Results: Subjects (n=11, 4 female, 7 male) had a median age of 48 years (range 21–72); four had suspected lymphoma pre-splenectomy, while 7 had a prior diagnosis and had splenectomy for diagnostic (to rule out transformation) or other purposes. Median time between PET and splenectomy was 1.25 months. Of 5 patients with low metabolic activity on PET; three had benign findings at splenectomy and two had splenic involvement of previously known mantle cell lymphoma. Three patients had intermediate metabolic activity on PET; all subsequently demonstrated marginal zone lymphoma at splenectomy. Three patients had high metabolic activity on PET; all were found to have diffuse large B cell lymphoma at splenectomy.

Conclusions: This study to our knowledge comprises the largest series to evaluate splenic FDG-PET uptake in patients with suspected or known lymphoma, followed by subsequent pathologic confirmation. Patients with low splenic SUVs appear to be less likely to have splenic involvement of lymphoma, while intermediate and high values may correlate with lymphoma histology. Our findings support a potential role for FDG-PET as a tool for use, in conjunction with clinical and laboratory assessment, in consideration of the need for splenectomy in these settings.
Banoxantrone (AQ4N), a Tissue Targeted Prodrug: Results of a Phase 1 Study in Lymphomas.
Authors: Furman Richard R. , Bartlett Nancy L. , Wong Alvin F. , McCulloch Leanne M. , Lam Gilbert N. , Rosenthal Todd , Flinn Ian , Leonard John P.
Published: October 2019

AQ4N is a prodrug which is selectively bioreduced by cytochrome P450 to AQ4, a potent DNA intercalator and topoisomerase II inhibitor. Preclinical studies demonstrate AQ4N selectively targets lymphoblastoid cell lines and hypoxic tumors. This study assessed the maximum tolerated dose (MTD), and pharmacokinetics (PK) of repeated dosing of AQ4N in patients (pts) with lymphoid malignancies. AQ4N was administered IV on Day 1 of a 21 day cycle to cohorts of at least three pts at doses of 400, 800, or 1200 mg/m2 for a maximum of 8 cycles. The dose was escalated as long as <33% of pts did not experience a dose limiting toxicity (DLT). 11 pts were enrolled with 3 pts treated at 400 mg/m2 and 4 pts treated at 800 and 1200 mg/m2 each. No pts experienced a DLT and no clinical MTD was identified. No further dose escalation was investigated due to reaching known maximum AQ4N solution solubility. The most common related adverse events (AE) observed were expected transient skin discoloration (100%), transient chromaturia (36%) and lymphopenia (27%), as well as fatigue (27%) and nausea (27%). AEs were primarily mild (Grade 1–2) with the exception of Grade 3 lymphopenias (n=3) and Grade 3 neutopenia (n=1) events. No dose reductions or dose delays resulted from these hematologic decreases. 6 pts experienced at least one serious AE, including: pneumonia, staph aureus bacteremia, acute respiratory distress syndrome (ARDS), dyspnea, and pleural effusion, none of which were attributed to AQ4N. The PK was linear over all doses studied. At 1200 mg/m2 (n=4), the Day 1 AQ4N Cmax was 122.3 ± 13.1 μg/mL, AUC0–∞ was 340.8 ± 68.7 μg·h/mL, and T1/2 was 3.2 h (range 2.8 to 4.1 h). One pt with follicular lymphoma dosed at 1200 mg/m2 had a partial response after the 4th cycle using the NHL standardized response criteria. The pt went on to complete all 8 cycles and to date remains in partial response. The bioreductive prodrug, AQ4N, is well-tolerated when administered on a repeated 21-day schedule at doses up to and including 1200 mg/m2. Further dose escalation was precluded since the known maximum solubility of AQ4N was reached. Blood levels of AQ4N achieved in this study appear to be within the range of potentially therapeutic levels of the active drug, AQ4, as seen in previous preclinical and clinical studies of solid tumors (Harris PA et al, 2006; Albertella MR et al., 2006). Preliminary evidence of anti-tumor activity was seen in one pt with follicular lymphoma. Further clinical studies of AQ4N administered both as a monotherapy and in combination with chemo- and radiation therapy are planned in B-cell neoplasms and solid tumor malignancies.
Prolonged Hematologic Toxicity from the Hyper-CVAD Regimen; Manifestations, Frequency, and Natural History in a Cohort of 125 Consecutive Patients.
Authors: Gill Saar , Lane Steven , Crawford Julie , Mollee Peter , Marlton Paula , Cull Gavin , Prince Miles , Seymour John Francis
Published: October 2019

Hyper-CVAD is a dose-intensive regimen which has been employed in the last decade to treat patients with a range of hematologic malignancies, with impressive efficacy. This regimen has been associated with considerable short-term hematologic toxicity, which has been mostly described as transient and reversible. However, recent reports have highlighted a concerning early rate of MDS or AML on medium-term follow-up. While cases of MDS/AML may be the most extreme end of the spectrum of hematopoietic stem cell damage, in the current retrospective study we describe other prolonged hematologic sequelae of this regimen. All patients were previously untreated and received Hyper-CVAD for the treatment of NHL or ALL. 125 patients (pts) with a median age of 47 (range, 15–76) were followed up for a median of 23 months (range, 1–84). The median number of Hyper-CVAD cycles was 6 (range, 2–9). Follow-up for blood counts was censored at the next cytotoxic therapy. At 3 months post therapy, 78 patients were evaluable. Thirty-two (41%) had achieved full blood count recovery and 45 (59%) had persisting cytopenias. Dose intensity delivered was significantly associated with persisting cytopenias (p = 0.028), but age (p = 0.61), underlying disease (0.49) and bone marrow involvement at diagnosis (p = 0.81) were not. Of the patients who had not normalised all counts by 3 months, anemia was present in 56% (median 106 g/L, range 76–119), neutropenia in 47% (median 1.27 × 109/L, range 0.03–1.75) and thrombocytopenia in 76% (median 96 × 109/L, range 9–120). One, two and three lineage cytopenias were present in 42%, 42% and 16% of pts, respectively. The median time to normalisation of counts for those with post-treatment cytopenias in the respective lineages was 9 months (range, 6–12) for Hb, 6 months (range, 6–30) for neutrophils, and 6 months (range, 6–30) for platelets. The median time to normalisation of the lowered counts was 9 months (n = 18) in those with multi-lineage cytopenias and 6 months (n = 12) in those with one lineage cytopenia. At 1 year following Hyper-CVAD with no further therapy, 49 patients were evaluable and 3 (6%), 7 (14%), and 9 (18%) still had anemia, neutropenia or thrombocytopenia respectively. MDS/AML was diagnosed in 3 patients at 4, 21, and 21 months after therapy. Of these patients, one received an autologous stem cell transplant 2 months after Hyper-CVAD, one was cytopenic at 3 months, and one was lost to follow-up until re-presenting with AML. These results indicate a considerable rate of prolonged hematologic toxicity after Hyper-CVAD, and a minor rate of MDS at this limited follow-up. These findings likely reflect cumulative damage to hematopoietic stem cells.
Improvement of Overall Survival in Mantle Cell Lymphoma during the Last Decades.
Authors: Herrmann Annina , Hoster Eva , Dreyling Martin , Unterhalt Michael , Brittinger Günter , Hiddemann Wolfgang
Published: October 2019

Purpose: Mantle cell lymphomas (MCL) represent a lymphoma subtype with an especially poor long term prognosis. Although various therapy strategies have been applied within the last 30 years, MCL is still considered incurable by conventional chemotherapy.

Patients and methods: To determine whether patient outcome has improved during the last decades, we compared data from the “Kiel lymphoma group” (KLG) collected in the years 1975–1986 with data from the “German lymphoma study group” (GLSG) of the years 1996–2004. To minimize potential heterogeneity of risk profiles of the two patient populations, frequency matching was pursued. Additionally, the effects of other potential prognostic parameters were adjusted by a Cox proportional hazard model.

Results: A total of 520 patients were evaluable for analysis. The median overall survival significantly increased from 2.7 years up to 4.8 years (p<0.0001; hazard ratio (HR) 0.44) and 5-year-survival of advanced MCL more than doubled from 22% [95% CI, 13% to 31%] to 47% [95% CI, 38% to 55%]. Multivariate analysis identified a poor performance status (HR 2.26), a high LDH level (HR 1.75), and age (HR 1.38) as additional prognostic variables.

Conclusion: Patients with advanced MCL benefit from the progress of medical treatment resulting in almost doubling of overall survival during the last 3 decades, even though relapses are regularly observed.
Phase II Trial of the Bonn Polychemotherapy Protocol with Deferred Radiotherapy in Patients with Primary Central Nervous System Lymphoma.
Authors: Schmidt-Wolf Ingo G.H. , Juergens Annika , Pels Hendrik , Glasmacher Axel , Schulz Holger , Engert Andreas , Linnebank Michael , Reichmann Heinz , Kroschinsky Frank , Vogt-Schaden Marlies , Egerer Gerlinde , Fliessbach Klaus , Schlegel Uwe
Published: October 2019

Objectives: The trial was performed to evaluate response rate, response duration, overall survival, and toxicity in primary central nervous system lymphoma (PCNSL) after systemic and intraventricular chemotherapy with deferred radiotherapy.

Patients and Methods: From 09/1995 to 12/2002, 88 patients with PCNSL (median age 62 years) were enrolled onto a pilot/phase II study evaluating chemotherapy without radiotherapy. A high-dose methotrexate (MTX) (cycles 1,2,4,5) and cytarabine (ara-C) (cycles 3,6) based systemic therapy (including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide) was combined with intraventricular MTX, prednisolone and ara-C. Primary endpoint was time to treatment failure (TTF), secondary endpoints were response, overall survival, response duration, 5-year-survival fraction and (neuro)toxicity.

Results: Eighty-four of 88 patients were evaluable for response. Of these, 46 (54%) achieved complete response (CR), 4 (5%) complete response/unconfirmed, 8 (10%) partial response (PR), and 14 (17%) progressed under therapy. Seven (8%) out of 84 patients died due to treatment-related complications. In five (6%) of 84 patients therapy had do be discontinued due to severe treatment related complications. Follow-up is one to 124 months (median 42 months). Kaplan Meier estimates for median time to treatment failure (TTF), median overall survival and median response duration are 19 months, 55 months and 37 months respectively. For patients aged 60 years or older, the respective numbers were 9 months, 34 months and 24 months; in patients younger than 60 years Kaplan Meier estimate for TTF is 49 months, median overall survival and median response duration have not been reached yet. The 5-year survival fraction is 72% in patients < 60 years and 24% in older patients. Systemic toxicity was mainly hematologic. Ommaya reservoir infection occurred in 20 (23%) patients. In a subgroup of patients (n=23) treated with chemotherapy alone serial neuropsychological testing showed no chemotherapy related cognitive decline in any patient. However, 8 out of 13 documented elderly patients treated with cranial irradiation as salvage therapy developed severe cognitive deficits.

Conclusions: Primary chemotherapy based on high-dose MTX and ara-C is highly efficient in PCNSL. A substantial fraction of patients < 60 years can obviously be cured with this regimen.
SGN-70, a Humanized Anti-CD70 Antibody, Targets CD70-Expressing Hematologic Tumors.
Authors: McEarchern Julie A. , McDonagh Charlotte F. , Smith Leia M. , Klussman Kerry , Oflazoglu Ezogelin , Morris-Tilden Carol , Stone Ivan , McCormick Renee , Carter Paul , Gerber Hanspeter , Grewal Iqbal S. , Law Che-Leung
Published: October 2019

Antigens expressed on malignant cells in the absence of significant expression on normal tissues are highly desirable targets for therapeutic antibodies. CD70 is a TNF superfamily member whose normal expression is restricted to activated lymphocytes but is aberrantly expressed in hematologic malignancies and solid tumors including non-Hodgkin’s lymphoma (NHL), Hodgkin’s disease, multiple myeloma (MM), Waldenstrom’s macroglobulinemia, renal cell carcinoma, glioblastoma, and nasopharyngeal carcinoma. To target CD70-expressing hematologic malignancies, we have engineered a humanized IgG1 anti-CD70 antibody that mediates lysis of tumor targets via ADCC and CDC and facilitates antibody dependent cellular phagocytosis in vitro. In vivo, administration of SGN-70 prolonged survival of SCID mice bearing CD70+ disseminated human NHL or MM xenografts. Intravenous injection of the CD70+ MM cell line MM.1S resulted in disease as measured by onset of paralysis, presence of CD138+ MM cells in the bone marrow, and increasing levels of circulating human Ig lambda light chain. SGN-70 treatment of MM.1S-bearing mice significantly delayed onset of paralysis and reduced the monoclonal protein levels detected in serum approximately 4-fold compared to untreated or non-binding antibody control-treated mice. Whereas myeloma cells comprised 32±6.4 % of mononuclear cells in the long bone marrow of control mice, SGN-70 treatment reduced the myeloma cell fraction to 4.9±1.9% of mononuclear cells recovered. SGN-70 treatment also significantly extended the survival of mice bearing disseminated Raji tumors (NHL) compared to control mice. Survival benefit was absent when mice received an Fc-modified antibody deficient in effector functions, confirming that the activity of SGN-70 in these models was dependent upon Fc-FcγR interaction with host immune cells. Together, these data demonstrate that SGN-70 possesses effector cell-mediated antitumor activity and provides rationale for clinical study of SGN-70 in CD70+ hematologic malignancies.
Inhibiton of mTOR by RAD001 Restores Normal B Cell Differentiation and Prevents B Cell Lymphomas in Eμ-MYC Mice.
Authors: Wall Meaghan , Poortinga Gretchen , Cardozo Daniela , Johnstone Ricky W. , McArthur Grant A.
Published: October 2019

The c-Myc proto-oncogene encodes a bHLH-LZ transcription factor that regulates proliferation, differentiation and apoptosis. Deregulated expression of c-MYC is a frequent finding in a wide variety of human cancers, including B cell lymphoma. One emerging function of c-MYC is the regulation of ribosome biogenesis, protein synthesis and metabolism i.e. cell growth. mTOR, a key downstream signal transduction molecule in the PI3K/AKT growth regulatory pathway, is amenable to pharmacological inhibition by rapamycin analogues such as RAD001. We hypothesized that control of cell growth by c-MYC is important for its ability to regulate differentiation and act as an oncogene and that RAD001, by limiting cell growth, would attenuate the transforming properties of c-MYC. In Eμ-myc mice the c-myc transgene is under the control of the immunoglobulin heavy chain enhancer (Eμ). Constitutive expression of c-MYC results in a polyclonal expansion of B cell precursors followed by lymphoma development. In the current study ‘pre-lymphomatous’ Eμ-myc mice were randomized to receive RAD001 5mg/kg (n=20) or placebo (n=18) 6 days per week from 4 weeks of age. Peripheral blood B cells were analyzed by surface marker expression after 2, 4 and 8 weeks of therapy. Mice were monitored weekly for the development of lymphadenopathy. 2 weeks of treatment with RAD001 significantly reduced the numbers of B cells in the blood of Eμ-myc mice compared to placebo (1.37±0.13 ×103/μL in the RAD001 arm versus 3.41±0.64 ×103/μL in the placebo arm, p<0.05). In particular, there was preferential suppression of less mature circulating B cell precursors over mature B cells by RAD001 resulting in B cell developmental subset profiles more closely approaching those of wild-type mice (Figure 1). Treatment with RAD001 was associated with improved lymphoma-free survival; 13/14 lymphoma-free mice (92.9%) versus 6/11 (54.6%) in the placebo group in an interim analysis of mice that had received at least 60 days of therapy. These results indicate that RAD001 can firstly oppose the expansion of B cell precursors and secondly reduce the incidence of malignant transformation induced by deregulated expression of c-MYC in B lymphocytes. These findings have implications for the application of mTOR inhibitors in the treatment or prevention of malignancies associated with MYC.

Figure 1. B cell subsets after weeks of therapy Figure 1. B cell subsets after weeks of therapy
Interleukin-21-Induced Apoptosis and Cell Death of Diffuse Large B-Cell Lymphoma (DLBCL) Cell Lines and Primary Tumors Are Associated with an Induction of Bim.
Authors: Sarosiek Kristopher A. , Chen Jun , Pham Dien G. , Nechushtan Hovav , Avisar E. , Kindsvogel Wayne , Lossos Izidore S.
Published: October 2019

IL-21, a member of the IL-2 cytokine family, is reported to have an immune-mediated anti-tumor activity against renal cell carcinoma, malignant melanoma and Non-Hodgkin’s Lymphoma (NHL) cell lines in xenograph animal models. Whether IL-21 exhibits direct anti-tumor activity against NHL cell lines and primary tumors is presently unknown.

We analyzed seven DLBCL and two Burkitt’s lymphoma cell lines for IL-21 receptor (IL-21R) expression. IL-21R was expressed at high levels in the two Burkitt’s lymphoma cell lines (RAJI and RAMOS). Out of the seven DLBCL lines, four expressed high levels of the receptor (OCI-LY-3, OCI-LY-7, OCI-LY-19, and RCK-8) while three had low receptor expression levels (OCI-LY-10, SU-DHL-4, and SU-DHL-6). IL-21 stimulation induced tyrosine phosphorylation of STAT-1, -3, and -5 as early as fifteen minutes post treatment in DLBCL lines expressing either high (OCI-LY-3 and RCK-8) or low (SU-DHL-6 and OCI-LY-10) levels of IL-21R.

In six of the seven DLBCL lines tested, IL-21 dramatically inhibited or completely abolished cellular proliferation at 25 ng/mL and 100 ng/mL concentrations, respectively. In contrast, one DLBCL cell line (OCI-LY-3) exhibited a threefold increase in cellular proliferation after stimulation with 25 ng/mL IL-21, but proliferation was effectively inhibited by 100 ng/mL IL-21.

Marked apoptosis and cell death were observed by flow cytometry at 72 hours post IL-21 exposure in all nine NHL cell lines tested with the exception of OCI-LY-3 which exhibited no significant change in cell viability. The IL-21-induced apoptosis was associated with an activation of caspases 3/7, 8, and 9, detected as early as 12 hours post IL-21 exposure. IL-21 also induced an increase in the levels of the pro-apoptotic protein Bim in all the cell lines exhibiting marked apoptosis. In contrast, Bim protein levels decreased in response to the IL-21 stimulation in the resistant OCI-LY-3 DLBCL cell line. IL-21 treatment led to a decrease in the protein levels of Bcl-2 in all the analyzed cell lines except OCI-LY-3. An increase in expression of Bcl-6 protein in three of the four tested DLBCL cell lines was observed in response to IL-21 exposure.

In primary tumors, IL-21 induced apoptosis in two of two DLBCLs, two of three follicular lymphomas, and two of six chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes or in the HeLa or 293T control cells. These results suggest that IL-21 exhibits a direct anti-tumor effect on the DLBCL cell lines and primary tumors and point to a potential applicability of IL-21 in anti-DLBCL therapy. Further work interrogating the molecular mechanism of the IL-21-induced apoptosis of DLBCL cells in vitro and in animal models is in progress.
Activity of Bendamustine (TREANDA™) in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma Cells with Alterations in DNA Damage Response Pathway.
Authors: Roué Gaël , López-Guerra Mónica , Milpied Pierre , Pérez-Galán Patricia , Villamor Neus , Campo Elias , Colomer Dolors
Published: October 2019

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are two different types of mature B-cell non-Hodgkin’s lymphoma (NHL). CLL has an indolent natural history and patients are very responsive to frontline chemotherapy. Unfortunately, multiple relapses are inevitable, and ultimately, no regimen or treatment strategy offers a distinct survival benefit over another. In contrast, patients with MCL generally experience a more aggressive course, with rapid disease progression and also without specific therapeutic options. Bendamustine hydrochloride (Treanda™) is a multifunctional, alkylating agent that exhibits single-agent activity in multiple hematologic and solid tumors. Recently, the combination of bendamustine with rituximab has demonstrated to be a highly active regimen in the treatment of low-grade lymphomas and MCL. However, very little is known about its mode of action. The ability of bendamustine to induce apoptosis in vitro in MCL and CLL cells and the mechanisms implicated in bendamustine-evoked cell death signaling were investigated. Bendamustine exerted cytostatic and cytotoxic effects in 11 MCL cell lines and primary tumor cells from 7 MCL patients and 10 CLL patients independent of their p53 status, and other gene alterations. In vitro treatment of cells with bendamustine induced activation of both p53-dependent and -independent signaling pathways that converged in all cases to the activation of the pro-apoptotic protein Noxa, conformational changes of Bax and Bak, and mitochondrial depolarization. These events led to cytosolic release of the mitochondrial apoptogenic factors cytochrome c, Smac/DIABLO and AIF, and activation of both caspase -dependent and -independent cell death. Genotoxic stress and caspase-independent cell death are often associated with the generation of reactive oxygen species (ROS). We observed that ROS production was a key step in the induction of apoptosis by bendamustine, since pre-incubation of tumor cells with ROS scavengers reverted all the typical hallmarks of apoptosis. Furthermore, bendamustine exerted a cytotoxic effect in p53 deleted CLL cases that were resistant to fludarabine treatment. These findings support the use of bendamustine as a therapeutic agent in MCL and CLL cells and also establish the basis for the use of bendamustine in lymphoid malignancies that show resistance to classic genotoxic agents that depend on cellular p53 status.
Antibody-Drug Conjugates Targeted to CD79 for the Treatment of Non-Hodgkin’s Lymphoma.
Authors: Polson Andrew G. , Yu Shang-Fan , Elkins Kristi , Zheng Bing , Clark Suzanna , Ingle Gladys , Slaga Dion , Giere Lynne , Du Changchun , Stephan Jean-Philippe , Young Judy , Vandlen Richard , Chang Wesley , Scales Suzie , Ross Sarajane , Eaton Dan , Ebens Allen
Published: October 2019

Non-Hodgkin’s lymphoma (NHL) is an attractive indication for the use of antibody-drug conjugate (ADC) therapeutics since NHL is responsive to conventional chemotherapeutic and antibody treatments. Additionally, experience with rituximab indicates that depletion of normal B-cells can be tolerated without untoward side effects, suggesting that B-cell specific surface proteins that are also prevalent in NHL are potential ADC targets. ADCs with stable linkers have lower toxicity than ADCs containing the same drugs with less stable linkers, however, for these ADCs to be effective the antibody must be degraded to release the active drug. This limits potential targets to surface antigens that upon antibody binding are highly internalized and subsequently degraded. We hypothesized that components of the B-cell receptor (BCR) would be excellent targets for stable-linker ADCs since, when cross-linked, the BCR is targeted to the lysosomal-like compartment MIIC as part of B-cell antigen presentation. In particular, CD79, the signaling component of the B-cell receptor, comprised of two peptide chains CD79a (Igα, mb-1) and CD79b (Igβ, B29), seemed a promising target for ADC treatment of NHL. CD79’s expression is restricted to the B-cell lineage and is on the surface of almost all NHLs. Unlike surface-Ig targeted therapies, which require a new drug for each cancer clone, CD79 sequences are invariant and a single drug is appropriate for the entire population. We found that ADCs targeted to CD79b were more effective than those targeted to CD79a. Furthermore, excellent efficacy is demonstrated with ADCs containing stable linkers and our data indicate in particular, that both anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are effective at low doses in subcutaneous cell-line xenograft models of follicular lymphoma, mantle cell lymphoma, Burkitt’s lymphoma and disseminated Burkitt’s lymphoma. Mechanism-of-action experiments show that anti-CD79b antibodies down regulate surface BCR expression and internalized antibody accumulates in the lysosomal-like MIIC compartment as hypothesized. We evaluated a number of ADCs targeted to other B-cell antigens and found that they were not effective if the ADC had a stable linker, indicating that CD79 has special properties as a target for ADCs. Our results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are particularly promising therapeutics for the treatment of NHL.
Nano-Fluidic Detection of Oncoprotein Signaling in Preclinical and Patient Lymphoma Samples.
Authors: Fan Alice C. , Deb-Basu Debabrita , Horoschak Melissa , Shirer Amy , Voehringer David , O’Neill Roger , Felsher Dean W.
Published: October 2019

Inactivation of oncogenes can be an effective cancer therapy. Determining precise levels of oncogene expression is important in the development of drugs to target oncogenes. We have developed a novel automated nano-fluidic Western-blot-like technology to detect and quantify oncogene expression in small numbers of mouse and human hematopoietic tumor cells. To detect different levels of oncogene expression, we generated transgenic mice in which the MYC or BCL2 oncogenes are regulated conditionally via the Tetracycline Regulatory System (Tet-system). Using lymphoma- derived cell lines from these mice, we titrated the level of oncogene expression by adding different concentrations of doxycycline in vitro. We were able to distinguish among different levels of oncogene expression in cell lysates, with high sensitivity in as few as 400 cells by nano-fluidic detection. Next, lymphoma derived cell lines were injected subcutaneously into syngeneic mice. Upon tumor development, MYC or BCL2 oncogenes were inactivated in vivo. Both MYC and BCL2 levels decreased in serial fine needle aspirations (FNAs) of tumor nodules upon parallel analysis with nano-fluidic detection and Western blot. Finally, we used nano-fluidic detection to determine levels of MYC, BCL2, AKT and ERK in lymph node samples from patients with follicular, transformed DLBC, Burkitt’s, and mantle cell lymphomas. BCL2 was overexpressed in mantle cell and follicular lymphoma patients, confirmed by Western analysis, whereas MYC was found to be overexpressed in Burkitt’s lymphoma. These results demonstrate that nano-fluidic detection technology may be used both as a preclinical tool for the assessment of changes in oncoprotein signaling and as a clinical diagnostic modality on microscopic clinical specimens.
Triterpenoids Display Single Agent Activity in a Mouse Model of CLL/SBL.
Authors: Kress Christina L. , Konopleva Marina , Krajewska Maryla , Martinez-Garcia Vanesa , Lefebvre Sophie , Hyer Marc L. , McQueen Teresa , Andreeff Michael , Reed John C. , Zapata Juan
Published: October 2019

The synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) induces apoptosis of leukemia cells through a novel mechanism and has recently entered Phase I human clinical trials. We studied the effects of CDDO and its imidazolide derivative (CDDO-Im) on cultured human chronic lymphocytic leukemia (CLL) cells and on B cells from TRAF2DN/Bcl-2 transgenic mice, a new mouse model of CLL and small B cell lymphoma (SBL). Both triterpenoids efficiently induced death of malignant human and mouse B-cells ex vivo, although CDDO-Im was over 10-fold more potent than CDDO. Treating TRAF2DN/Bcl-2 mice that had developed leukemia with liposome-formulated CDDO or CDDO-Im resulted in significant reductions of B cells in blood, spleen and lung, with CDDO-Im more potent than CDDO, while treatment with empty liposomes had no impact on disease. Analysis of blood cells recovered from treated mice showed that CDDO-Im is a potent inducer of cell dead in vivo. Furthermore, CDDO-Im efficiently eradicated mouse CLL cells but had a lesser effect on the viability of normal B cells. These results demonstrate that triterpenoids CDDO and CDDO-Im reduce leukemia and lymphoma burden in vivo in a transgenic mouse model of CLL/SBL and suggest that CDDO-based synthetic triterpenoids should be tested for clinical activity in patients with CLL. Our results also provide evidence of the suitability of our mouse model of CLL/SBL as a preclinical platform for chemotherapeutic drug testing.
FLT3-ITD Mediated Growth Advantage of Human CD34+ PBPCs Is Dependent on Stromal Cells.
Authors: Jacobi Angela , Ryser Martin F. , Koch Sina , Brenner Sebastian
Published: October 2019

Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. In search for a valid model system for FLT3-ITD AML we greatly improved retroviral gene transfer of the mutated FLT3 receptor into human G-CSF mobilized CD34+ peripheral blood progenitor cells (PBPCs). In the past, retroviral vector production for FLT3 had been hampered by low virus titers related to transgene toxicity in the virus producing cells. Virus vector production in the presence of the FLT3 inhibitor GTP14562 strongly increased the virus vector titer. Subsequent ultracentrifugation and resuspension of the concentrated virus vector in fresh medium enabled us to consistently transduce CD34+ PBSCs to levels of greater than 50%. For individually tested CD34+ PBPC from 5 donors, co-cultivation of FLT3-ITD overexpressing PBPC on the irradiated mouse stromal cell line M210-B4 resulted in a 6 fold increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, GFP-transduced or FLT3-wildtype transduced PBPCs. The enhanced proliferation of the FLT3-ITD transduced cells was associated with decreased CD34 expression compared to control-transduced cells. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced PBPCs. In contrast, cultivating the same FLT3-ITD overexpressing PBPCs in liquid culture (X-VIVO10, 1%HSA, TPO, SCF, FLT3 and IL-3) resulted in a moderate or no growth advantage or in an even reduced cell viability (dependent on the donor PBPCs used), which indirectly highlights the influence of stromal cells on FLT3-ITD overexpressing PBPCs. And in contrast to previously published data from murine cells, addition of the CXCR4 ligand stromal cell derived factor-1 (SDF-1) to liquid cultures did not confer an additional growth advantage for FLT3-ITD positive cells. Using an improved transduction protocol we demonstrate that the robust FLT3-ITD mediated growth advantage for human PBPCs is dependent on co-cultivation on a stromal cell layer and we show that the enhanced proliferation can be partly inhibited by the CXCR4 inhibitor AMD3100. Studies to delineate factors that confer FLT3-ITD specific cell growth on stromal cells are under way.
Prospective Isolation of Mesenchymal Stem Cells from Human Bone Marrow and Placenta Using a Frizzled-9-Specific Monoclonal Antibody.
Authors: Battula Venkata L. , Treml Sabrina , Kanz Lothar , Buhring Hans-Jorg
Published: October 2019

We have recently shown that Frizzled-9 (FZD9) is expressed on the cell surface of multipotential mesenchymal stromal cells derived from human bone marrow (BM) and placenta (PL). To test whether FZD9 is also expressed on primary mesenchymal stem cells (MSC) with CFU-F (colony forming unit fibroblast) capacity, we used the FZD9-specific monoclonal antibody W3C4E11 to fractionate BM and PL cells into FZD9+ and FZD9- populations. Defined numbers of sorted cells were then cultured in a serum free bFGF-containing medium, originally designed fur the culture of human ES cells. 14 days after culture, the resulting fibroblastoid cells were stained with Giemsa staining solution and colonies consisting of > 50 cells were enumerated. In the FZD9+ fraction, a 70 fold increase of the CFU-F frequency in PL and a 80–90 fold increase in BM was observed. Cultured FZD9+ cells also expressed the stem cell markers Oct-4, nanog-3, and nestin, as well as SSEA-4, TRA1-2-49/6E, CD105, CD13, CD164, CD318, and W8B2B10 antigen. After culture under appropriate conditions, FZD9+ cells could be differentiated into cells of the ectodermal (neuronal like cells), endodermal (pancreatic like cells), and mesodermal (adipocytes and osteoblasts) lineages, indicating the multi-lineage differentiation capacity of these cells. In conclusion, FZD9 is a novel marker for the prospective isolation of MSC from human BM and PL.
Prospective Identification and Characterization of Human Mesenchymal Stem Cell Subpopulations.
Authors: Tormin Ariane , Brune Jan C. , Borg Åke , Neumann Ulf , Olofsson Tor , Lauster Roland , Jakobsen Sten-Eirik , Scheding Stefan
Published: October 2019

Mesenchymal stem cells (MSC) are multipotent cells that readily differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes. They support hematopoiesis (stroma function) and have potent immunomodulatory properties making them promising candidates for clinical use. Although MSC cultures are heterogeneous in morphology, no specific markers are available that would allow for the prospective isolation of distinct subpopulations. Therefore we investigated whether potential MSC subpopulation markers could be identified by gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics). Human MSC were generated in standard cultures and stained with carboxyfluoresceine-succinimidylesther (CFSE) for cell division tracking. FACS analysis of CFSE-stained cells after 10 days culture showed that the majority of cells had undergone more than 3 cell divisions (51.2 ± 1.7%) whereas only 3.5 ± 0.9 % had not divided. Based on these results MSC were sorted by FACS according to their divisional history into non-proliferating cells (NP-MSC) and rapidly-proliferating cells (RP-MSC). Interestingly, the CFU-F frequencies of the NP-MSC were considerably (5-times) lower compared to RP-MSC. Furthermore, RP-MSC with low forward-/side-scatter properties had a higher CFU-F frequency than those with high scatter properties. Comparative oligonucleotide microarray analysis of FACS-sorted NP-MSC and RP-MSC populations identified a total of 112 differentially (≥4-fold) expressed genes. Fifty genes were significantly higher expressed in RP-MSC with 12 of them being cell cycle associated. Sixty-two genes showed a higher expression in NP-MSC and more than 20 of them were related to adhesion molecules, extracellular matrix proteins or cell senescence. Two of the differentially expressed genes (VCAM-1, FMOD) that corresponded to cell surface molecules were further tested for their potential to prospectively identify MSC subpopulations. Remarkably, FACS analysis using anti-VCAM-1 (CD106) and anti-fibromodulin (fmod) antibodies showed that approximately 40% and 3% of MSC were CD106neg/fmodneg and CD106pos/fmodpos, respectively. MSC were then sorted by FACS according to their expression of these markers and assayed for CFU-F frequency. Significant differences in CFU-F frequencies were observed between the double-negative and the double-positive MSC (12.3 ± 2.8 and 4.7 ± 2.1 colonies per 100 cells for CD106neg/fmodneg and CD106pos/fmodpos MSC, respectively). Experiments to test for differentiation potential differences are ongoing. Taken together, the data clearly demonstrate functional differences between MSC subpopulations. Furthermore, cell sorting based on proliferation characteristics and gene expression profiling enabled to identify surface markers that allowed for the first time to prospectively identify MSC subpopulations.
Novel Small Molecule MDM2 Inhibitor MI-63 Induces p53-Dependent Apoptosis in AML Cell Lines.
Authors: Samudio Ismael , Dietrich Martin , Corn Paul , Yang Dajun , Borthakur Gautam
Published: October 2019

Although TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). We investigated the effects of MI-63, a small molecule that activates p53 by inhibition of MDM2-p53 interaction [ Ki value of 3 nM (J Med Chem.2006;49(12):3432–5)] in AML cell lines. Treatment with MI-63 triggered apoptosis (evidenced by loss of membrane potential and externalization of phosphatidylserine) in AML cell lines with wild-type p53 (OCI-AML-3 and MOLM13) in a time and concentration-dependent manner (IC50 at 72 hrs.= 2.5 μM for OCI-AML-3 and 1 μM for MOLM-13), while a p53-null AML cell line (HL-60) was resistant (IC50 not reached at 10 μM). Moreover, knockdown of p53 in OCI-AML3 cells rendered this cell line resistant to MI-63 induced apoptosis while control vector infected OCI-AML-3 cells remained as sensitive to MI-63 similar to the parental cells.

Mechanistic studies showed that MI-63 blocks G1/S phase transition in AML cells with wild-type p53 resulting in accumulation of cells in G1 phase (percentage cells inG1 phase at 24 hrs. = 88.66% vs 43.49% in cultures with DMSO control) while MI-61, a skeletally related but inactive control compound failed to do so (41.63%). Treatment with MI-63 increased cellular levels of p53 and p53 dependent proteins in OCI-AML-3 cells that include p21 and BH3-only pro-apoptotic protein Puma and pro-apoptotic multi-domain Bcl-2 family member Bax. Additionally, MI-63 induced a profound decrease in the levels of MDM4, an MDM2 homolog that has been reported to mediate resistance to the effects of nutlin-3a, suggesting that MI-63 may offer a therapeutic advantage in cells expressing high levels of MDM4.

Finally, supporting the concept that increased levels of p53 modulate the apoptotic rheostat both directly, by behaving as a BH3-only protein, and indirectly by increasing the levels of sensitizer BH3-only proteins, MI-63 potently synergized with AT-101, an orally available pan inhibitor of Bcl-2, Bcl-xL and Mcl-1 (currently being evaluated as an antitumor agent in Phase I/II trials by Ascenta Therapeutics), to induce mitochondrial dysfunction and apoptosis in OCI-AML-3 cells (average combination index = 0.055±0.019).

Taken together our results support preclinical evaluation of novel small molecule MI-63 alone and in combination with Bcl-2 inhibitors for the therapy of AML. The studies in primary AML samples are ongoing.

Fig.1: MI-63 Induced Apoptosis Requires Intact p53 Fig.1:. MI-63 Induced Apoptosis Requires Intact p53 Fig.2: Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included) Fig.2:. Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)
Serotonin Receptor Antagonists Have an In Vitro and In Vivo Anti-Myeloma Effect That Is Mainly Mediated by Caspase Dependent Apoptosis.
Authors: Ocio Enrique M. , Hideshima Teru , Yasui Hiroshi , Tanyel Kiziltepe , Ikeda Hirosi , Okawa Yutaka , Vallet Sonia , Jin Janice , Ishitsuka Kenji , Raje Noopur , Catley Laurence , Anderson Kenneth C.
Published: October 2019

To date, serotonin (5-hydroxytryptamine: 5-HT) has been associated with the regulation of several central nervous system functions such as mood, temperature, memory, sleep or appetite. Importantly, a proliferative effect of 5-HT in both normal and tumoral tissue has recently been described. Therefore, serotonin-receptor antagonists have been tested for antineoplastic purposes. In this study, we have investigated the in vitro and in vivo anti-multiple myeloma (MM) effect of two analogs of 5-HT-receptor antagonists (ICI-735 and ICI-685). Both compounds induced cytotoxicity (MTT) and a decrease in proliferation (thymidine uptake) in several MM cell lines both sensitive and resistant to conventional therapies, as well as freshly isolated MM cells, with an IC-50 at 24h ranging between 3-10 μM for ICI-735 and 5–20 μM for ICI-685. No significant toxicity was observed at these doses of both drugs in PBMCs obtained from normal donors. In order to analyze the efficacy of the drugs in the presence of the bone marrow (BM) microenvironment, we tested both compounds in MM1S cells co-cultured with IL-6, IGF-1 or long-term patient BM stromal cells (SCs). ICI-735 and ICI-685 overcame the proliferative advantage conferred by the cytokines and BMSCs. Apoptosis was the main mechanism of cytotoxicity, assessed by Annexin-V staining. Treatment with ICI-735 and ICI-685 induced cleavage of PARP and caspase 3, 7, 8 and 9 at early time points, suggesting involvement of both extrinsic and intrinsic pathways of apoptosis. The presence of caspase-induced apoptosis was confirmed since a pan-caspase inhibitor (ZVAD-FMK) partially abrogated apoptosis, as well as the cleavage of PARP and caspases, induced by these compounds. An increase of AIF was also detected in the cytoplasmic fraction of cells treated with ICI-735, indicating a role for caspase-independent apoptosis. The in vivo efficacy was next evaluated in a xenograft murine (CB-17 SCID-mice) model. Five mice were injected intraperitoneally with 20 mg/Kg of ICI-735, while 5 received vehicle alone. No significant toxicity was detected, except for a slight somnolence after the first doses, in the mice that received the drug. A decrease in tumor volume was observed in the treated group and, despite the small number of mice, survival analysis showed a trend toward prolonged survival for mice receiving the drug (p=0.11). Ongoing studies are providing the preclinical rationale for clinical protocols to improve patient outcome in MM.