B‐cell frequencies and immunoregulatory phenotypes in myeloproliferative neoplasms: Influence of ruxolitinib, interferon‐α2, or combination treatment

Introduction. Secondary malignancies, particularly solid tumors, are common in patients with chronic lymphocytic leukemia (CLL), but association of myeloproliferative neoplasms and chronic lymphocytic leukemia in the same patient is very rare. Case Outline. We report of a 67-year-old man with B-cell chronic lymphoid leukemia (B-CLL) who developed primary myelofibrosis (PMF) nine years after initial diagnosis. Patient received alkylation agents and purine analogue, which can be a predisposing factor for the development of myeloproliferative neoplasms. JAK2V617F mutation was not present initially at the time of CLL diagnosis, but was found after nine years when PMF occurred, which indicates that B-CLL and PMF represent two separate clonal origin neoplasms. Conclusion. Pathogenic mechanisms for the development of myeloproliferative and lymphoproliferative neoplasms in the same patient are unknown. Further research is needed to determine whether these malignancies originate from two different cell clones or arise from the same pluripotent hematopoietic stem cell.

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PF669 RUXOLITINIB TREATMENT AND RISK OF B-CELL LYMPHOMAS IN MYELOPROLIFERATIVE NEOPLASMS

HemaSphere ◽

10.1097/01.hs9.0000560960.68270.0b ◽

2019 ◽

Vol 3 (S1) ◽

pp. 287-288

Author(s):

C. Cavalloni ◽

E. Rumi ◽

S. Zibellini ◽

E. Boveri ◽

R. Riboni ◽

...

Keyword(s):

B Cell ◽

Myeloproliferative Neoplasms ◽

B Cell Lymphomas

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Ruxolitinib treatment and risk of B‐cell lymphomas in myeloproliferative neoplasms

American Journal of Hematology ◽

10.1002/ajh.25489 ◽

2019 ◽

Cited By ~ 4

Author(s):

Elisa Rumi ◽

Silvia Zibellini ◽

Emanuela Boveri ◽

Chiara Cavalloni ◽

Roberta Riboni ◽

...

Keyword(s):

B Cell ◽

Myeloproliferative Neoplasms ◽

B Cell Lymphomas

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Galiximab (anti-CD80 mAb)-Induced Cell Triggering Results in the Inhibition of Constitutive NF-Kb Activity in Raji: Contrasting Roles of Snail and RKIP in the Regulation of Drug/Immune Resistance

Blood ◽

10.1182/blood.v112.11.3623.3623 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3623-3623

Author(s):

Melisa Martinez-Paniagua ◽

Mario I. Vega ◽

Sara Huerta-Yepez ◽

Stavroula Baritaki ◽

James R. Berenson ◽

...

Keyword(s):

T Cell ◽

Tumor Cell ◽

Phase I ◽

B Cell ◽

Molecular Mechanism ◽

Combination Treatment ◽

Metastasis Suppressor ◽

Drug Induced ◽

Variable Regions ◽

Induced Apoptosis

Abstract The CD80 antigen, also called B7.1, is the natural ligand for the T cell receptor CD28 and which maintains T cell and B cell adhesion. Galiximab (anti-CD80 mAb) is a primatized (human IgG1 constant regions and Cynomolgous macaque variable regions) mAb that binds CD80 on lymphoma cells and has been shown in vitro to inhibit tumor cell proliferation, upregulate apoptosis and induce ADCC. A phase I/II trial as single agent Galiximab with dose escalation demonstrated that it is well tolerated and produced modest clinical activity. Also, a phase I/II trial evaluated the combination of Rituximab and Galiximab in patients with relapse refractory follicular NHL. The combination produced an overall response rate of 66% with a median PFS of 12.4 months. We have reported that Galiximab sensitized Raji and IM-9 cells to drug-induced apoptosis. The present study extends these findings and examines the underlying molecular mechanism by which Galiximab sensitizes NHL cells to apoptosis by cytotoxic drugs. We hypothesized that Galiximab inhibits intracellularly cell survival anti-apoptotic pathways such as constitutively activated NF-kB, leading to sensitization to drug-induced apoptosis. We have used CD80-expressing Raji cells as a model for our studies. We demonstrate that following treatment of Raji with Galiximab (25–50 μg/ml) for 24 hours, cell lysates were assessed for various gene products of the NF-kB pathway by Western. There were significant downregulation of both p65 and phospho-p65, both IkB-α and phospho-IkB-α and downstream inhibition of Bcl-2 and BclXL and induction of Bak. In addition, there was a strong induction of the metastasis suppressor and immune surveillance cancer gene product Raf-1 kinase inhibitor protein (RKIP) and downregulation of the inactive and phosphorylated form of RKIP. The induction of RKIP by Galiximab was, in part, the result of NF-kB-induced inhibition downstream of the metastasis inducer and RKIP transcription repressor Snail. Galiximab also inhibited downstream both the Fas and DR5 transcription repressor Yin-Yang 1 (YY1) concomitantly with upregulation of Fas and DR5. These findings establish a molecular mechanism by which Galiximab sensitizes tumor cells to drug/immune-induced apoptosis via inhibition of NF-kB and Snail and induction of RKIP expression. We have previously reported that Rituximab modifies intracellular pathways including NF-κB and sensitizes B-NHL to apoptosis (Jazirehi and Bonavida, Oncogene, 24:2121, 2005). Thus, the combination treatment with Rituximab and Galiximab, through common and complementary mechanisms, may result in the reversal of CD20+/CD80+ B-NHL tumor cell resistance. The studies also suggest the potential combination treatment of Galiximab and non-toxic chemotherapeutic drug or immunotherapeutic drug (example: TRAIL) in the treatment of refractory CD80+ B cell malignancies.

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Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v112.11.886.886 ◽

2008 ◽

Vol 112 (11) ◽

pp. 886-886 ◽

Cited By ~ 1

Author(s):

Lapo Alinari ◽

Erin Hertlein ◽

David M. Goldenberg ◽

Rosa Lapalombella ◽

Fengting Yan ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Preclinical Model ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

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Combination Treatment with JAK2 and PI3K Inhibitors in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v120.21.180.180 ◽

2012 ◽

Vol 120 (21) ◽

pp. 180-180

Author(s):

Meng Ling Choong ◽

Christian Pecquet ◽

Shi Jing Tai ◽

Jacklyn WY Yong ◽

Vishal Pendharkar ◽

...

Keyword(s):

Bone Marrow ◽

Combination Treatment ◽

Kinase Inhibitors ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Pi3k Inhibitor ◽

Spleen Weight ◽

Pi3k Inhibitors ◽

Jak2 Inhibitor ◽

Jak2 Inhibitors

Abstract Abstract 180 Background and Aims. The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel. The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment. Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results. Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone. Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions. Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

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Mevalonate pathway inhibitors in the treatment of B-cell chronic lymphocytic leukemia.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.6561 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 6561-6561

Author(s):

Ravi Kiran Bobba ◽

Indira Benakanakere ◽

Smitha Bearelly ◽

Monica Arya ◽

Richard Sleigtholm ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Cell Viability ◽

B Cell ◽

Combination Treatment ◽

Mevalonate Pathway ◽

Chemotherapeutic Agents ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

6561 Background: B-cell chronic lymphocytic leukemia (CLL) cells are arrested in G0/G1 phase of the cell cycle and are resistant to programmed cell death, hypothesized to contribute to the resistance of CLL cells to standard chemotherapy with curative intent. Methods: Mec-2 cells and Wac-3 cells are CLL cells that have been shown to be resistant to fludarabine and rituximab. We tested a novel enzyme inhibitor’s ability to render CLL cells sensitive to fludarabine and rituximab. Results: BIBB515, a lanosterol synthase inhibitor, at a concentration of 10μM, was able to reduce the cell viability from 82% in controls to 65% after 72 hours. Fludarabine 10μM alone did not reduce the cell viability, 82 % in controls compared to 80%. BIBB515+ fludarabine treatment for 72 hours, at the dose of 10μM each decreased the cell viability to 37%. Cell proliferation by MTT assay was 0.66±0.010 in control compared to 0.37±0.01 in BIBB515+fludarbine and 0.21±0.01 in BIBB515+ fludarabine+ rituximab. There is a 68% down-regulation of cell proliferation using this treatment. There was a two fold induction of CD 20 with combination treatment, and BIBB515 treatment. The mechanism of cell death in the combination treatment of BIBB515 and fludarabine may be due to the up regulation of cell surface marker CD-20. WAC-3 is another CLL cell line that is sensitive to fludarabine, and resistant to rituximab. BIBB515 sensitizes WAC-3 cells to CD 20 antibody rituximab. There is a 68.7% decrease in cell proliferation with combination treatment of BIBB515 and rituximab. Proliferation of Mec-2 cells were inhibited by 60µM and 30µM terbinafine. Ro-48-8071, showed dose-dependent activity, alone or in combination to fludarabine was seen to induce cell death in Mec-2 cells. Fludarabine alone did not have any effect on these cells. Conclusions: Inhibitors of the mevalonate pathway make resistant CLL cells sensitive to current chemotherapeutic agents. Exploiting this mechanism could alter the current treatment regimens, leading to control of the disease in advanced stages by either inducing the leukemic cells to be static or to regress. This strategy may also limit the toxicities involved with chemotherapy.

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