bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of Proteasome Inhibitors Bortezomib and NPI-0052 Trigger In Vivo Synergistic Cytotoxicity in Multiple Myeloma.
Authors: Chauhan Dharminder , Singh Ajita , Brahmandam Mohan , Podar Klaus , Hideshima Teru , Schlossman Robert , Richardson Paul , Munshi Nikhil , Palladino Michael , Anderson Kenneth C.
Published: April 2020

Bortezomib (Velcade™), the first in class proteasome inhibitor is FDA approved drug for the treatment of relapsed and relapsed/refractory multiple myeloma (MM). However, as with other agents, dose-limiting toxicities and the development of resistance limits its long-term utility. Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in MM cells; and importantly, is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 + bortezomib-induced synergistic apoptosis is associated with:

activation of caspase-8, caspase-9, caspase-3, and PARP; induction of ER-stress response and JNK; inhibition of migration of MM cells and angiogenesis; suppression of chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteolytic activities; and blockade of NF-kappa B signaling.

Studies in a xenograft MM model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth. Importantly, analysis of resected xenografted tumors show that 30–40% proteasome inhibition of all three (CT-L, C-L and T-L) proteasomal activities is sufficient to trigger significant MM cell apoptosis, confirming both the sensitivity of MM cells to proteasome inhibition and the importance of inhibiting all three proteolytic activities to obtain maximum response. Immunohistochemical analysis of MM tumors excised from NPI-0052 + bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. The clinical observation that bortezomib therapy can be associated with toxicity and drug-resistance, coupled with our present preclinical findings demonstrating that low doses of bortezomib together with NPI-0052 trigger a potent anti-MM effect in vitro and in vivo, suggests the promise of combination treatment strategies to enhance anti-MM activity, reduce toxicity, overcome drug resistance, and improve outcome in MM patients. In addition to the above studies, data related to combination of NPI-0052 with lenalidomide (Revlimid™) and with histone deacetylase inhibitors such as MS-275, Tubacin or LBH589, will be presented.
Expression of V617F JAK2 in Mice Leads to MPD Mimicking Human ET, Idiopahtic Myelofibrosis, and PV.
Authors: Shide Kotaro , Shimoda Haruko K. , Kumano Takashi , Karube Kennosuke , Kameda Takuro , Oku Seido , Takenaka Katsuto , Suzuki Keiko , Kubuki Youko , Harada Mine , Shimoda Kazuya
Published: April 2020

An acquired JAK2 V617F mutation has been detected in up to 90% of patients with polycythemia vera (PV) and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, how a single mutation may be responsible for such different clinical phenotypes is unknown. Mice transplanted with bone marrow cells in which V617F JAK2 was retrovirally expressed developed PV-like features, but not ET or IMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated transgenic mice expressing V617F JAK2 driven by the murine H2Kb promoter. We established two lines. The expression of V617F JAK2 mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines. One line showed leukocytosis after 4 months of age, with a predominance of granulocytes. Among 43 mice, examined after 3 months of age, 8 (19%) showed polycythemia and 14 (33%) showed thrombocythemia. Two polycythemia cases also showed thromobocytosis. The other line showed extreme leukocytosis and thromobocytosis at one month of age. The leukocytosis progressed as the animals aged, but the thrombocytosis tended to resolve at 8 months. They showed anemia that means Hb value from 9 to 10 g/dL at one month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly with myeloid cell and megakaryocyte invasion was observed. We conclude that in vivo expression of V617F JAK2 results in ET-like, IMF-like, and PV-like disease.
JAK2 Genotyping (Real-Time Allelic Discrimination) Reveals Rare JAK2 V617F-Positive Erythroid Colonies in Pediatric Patients with Essential Thrombocythemia.
Authors: Pospisilova Dagmar , Veselovska Jitka , Pekova Sona , Horvathova Monika , Solna Renata , Cmejlova Jana , Cmejla Radek , Belickova Monika , Stary Jan , Divoky Vladimir
Published: April 2020

Childhood essential thrombocythemia (ET) is a very rare disease belonging to a group of Bcr-Abl negative myeloproliferative disorders. Our study involved 15 pediatric ET patients diagnosed at the age of 6–17 years. Monoclonal hematopoiesis was detected only in one patient, who is recently an adult (diagnosed at the age of 9). JAK2 V617F was detected in whole blood DNA by allele-specific (AS)-PCR and BsaXI restriction analysis. In our hands AS-PCR sensitivity reached the detection limit of one homozygous cell in 2000 normal cells. In addition, more sensitive AS-RT-PCR was performed on RNA isolated from granulocytes and platelets from peripheral blood. Only one patient was JAK2 V617F-positive as detected by these established protocols. In the clonal assay of hematopoietic progenitors, most patients showed hypersensitivity to erythropoietin (Epo) in vitro with the growth of Epo-independent erythroid colonies (EECs) in some of them. Over 300 myeloid colonies were picked and analyzed by real-time allelic discrimination assay for JAK2 V617F mutation run on a RotorGene 3000 instrument (Corbet Research, Australia) using fluorescently labeled LNA-modified probes. We have detected a few JAK2 V617F-positive EECs and CFU-GMs in 4 patients from our cohort. For example in one patient out of 7 analyzed EECs, one colony was homozygous and one heterozygous for the mutation. In the same patient out of 10 analyzed CFU-GMs one was homozygous for the mutation. In another patient out of 27 analyzed EECs only one was heterozygous. Some positive clones were confirmed by direct sequencing. Our data clearly show that some childhood ET patients posses JAK2 V617F-positive hematopoietic colonies derived from in vitro assays of committed hematopoietic progenitors. The rarity of the mutated subclones and the inability to detect them from peripheral blood using standardized protocols suggest either that the culture conditions without addition of Epo led to a great enrichment of the mutated colony growth or that the JAK2 V617F positive clones may be repressed in ET. The presence of JAK2 V617F clone in our cohort was not associated with any severe clinical symptoms. It would be interesting to follow up the patients for possible subsequent acquisition of JAK2 mutation as well as possible evolution of the disease to a clonal one or to polycythemia vera.

This work was supported by Ministry of Health of the Czech Republic (Grant NR9471-3), Ministry of Education, Youth and Sports of Czech Republic (projects MSN 6198959205 and MSN 6198959216).
Role of JAK2V617F during In Vitro PV Erythroid Progenitor Differentiation.
Authors: Nussenzveig Roberto , Gaikwad Amos , Manshouri Taghi , Dwyer Karen , Zhang Ying , Kantarjian Hagop , Verstovsek Srdan
Published: April 2020

The dominant gain-of-function mutation in the tyrosine kinase JAK2 (1849 G to T) has been detected in over 80% PV patients and in approximately half of ET or PMF patients. The mutation results in a non-synonymous amino acid substitution at position 617 (valine to phenylalanine) in the JH2 pseudo-kinase auto-inhibitory domain. Current models predict that expression of mutant JAK2V617F in patient hematopoietic stem cells leads to activation of signaling pathways important for proliferation and survival. Furthermore, experimental evidence using murine models, in which JAK2V617F is retrovirally over-expressed, display a phenotype that closely resembles PV, including progression to myelofibrosis. Previous reports have indicated that “in vitro” expansion of PV progenitors favors erythroid precursors with decreased JAK2V617F mutational burden (Exp. Hematol. (2007) 35(4): 587–95). Investigators in these studies, however, did not report changes in JAK2V617F frequency in sorted live erythroid progenitors after “in vitro” expansion. Furthermore, CD34+cells from PV patients cultured in the presence of 20% serum indicated an inverse relationship between JAK2V617F frequency and Epo dose (Blood (2006) 108(9):3128–34). However, these reports failed to correlate Epo dose, and JAK2V617F frequency, with live “in vitro” expanded purified erythroid progenitor cells. Using our published procedure (Exp. Hematol. (2007) 35(4): 587–95), we expanded erythroid progenitors from healthy volunteers and JAK2V617F positive PV patients (mutational burden ranging from 20–90%) in serum-free media supplemented with SCF, Tpo, and Flt3L (100 ng/mL each - week 1) followed by SCF (50 ng/mL), IGF1 (50 ng/mL), and Epo (0, 0.003, 0.03, 0.3, and 3 Units/mL - week 2). Progression of progenitor expansion, erythroid lineage commitment, live/dead cells, and JAK2 mutation frequency was followed by flow cytometry and real-time PCR. At the end of week 2, cells were harvested, stained with anti-CD71 (transferrin receptor), anti-CD235A (glycophorin A), and the Aqua Live/Dead cell reagent. Stained cells were fixed in 2% paraformaldehyde, analyzed, and sorted on a FACSAria flow cytometer. Live, expanded progenitor cells were sorted into CD235A positive and negative populations, and subsequently used for estimation of JAK2 frequency by quantitative real-time PCR (Exp. Hematol. (2007) 35(1): 32–38). Our results indicate expansion and erythroid commitment of PV progenitor cells in the absence of Epo correlates with the patients’ intrinsic JAK2V617F burden. Furthermore, irrespective of JAK2V617F frequency in purified peripheral blood granulocytes, an inverse correlation between Epo dose and mutation frequency in “in vitro” expanded purified erythroid progenitors was not observed in all patients examined.
Results from a Phase II Study of Lenalidomide Oral Monotherapy in Relapsed/Refractory Indolent Non-Hodgkin’s Lymphoma.
Authors: Witzig Thomas E. , Vose Julie M. , Moore Timothy D. , Reeder Craig B. , Cole Craig E. , Justice Glen , Kaplan Henry P. , Voralia Michael , Pietronigro Dennis , Takeshita Kenichi , Ervin-Haynes Annette , Zeldis Jerome B. , Wiernik Peter H.
Published: April 2020

Background: Indolent non-Hodgkin’s lymphoma (NHL) has a median survival as long as 10 years but is rarely cured. Lenalidomide (Revlimid®), an immunomodulatory drug, is approved in the US for treatment of myelodysplastic syndromes associated with a del[q5] cytogenetic abnormality and is approved both in the US and the EU for the treatment of relapsed/refractory multiple myeloma. Lenalidomide also has activity in chronic lymphocytic leukemia and cutaneous T-cell lymphoma.

Aim: This study was designed to assess the safety and efficacy of lenalidomide monotherapy in patients with relapsed/refractory indolent NHL.

Methods: Patients with relapsed/refractory indolent NHL with measurable disease ≥ 2cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology.

Results: Forty-three patients were enrolled in the study and included in this analysis. The median age was 63 (43–89) and 17 were female. Histology was small lymphocytic lymphoma [SLL] (n=18), follicular center lymphoma grades 1, 2 [FCL] (n=22), nodal marginal B-cell lymphoma (n=2) and extranodal marginal zone B-cell lymphoma of MALT type (n=1). Median time from diagnosis to lenalidomide treatment was 4.5 (0.4–24) years and median number of prior treatment regimens was 3 (1–15). Eleven of the 43 patients (26%) exhibited an objective response (2 complete responses (CR), 1 complete response unconfirmed (CRu), 8 partial responses (PR)), 15 had stable disease (SD), 13 had progressive disease (PD) and 4 were not evaluated. Responses included 4/18 SLL (22%) and 7/22 FCL (32%). Median time to response was 3.6 months (range 1.7–4.1). Progression free survival is 4.6 months for all patients, > 7.7 months (range 4.4 - > 13.5) for responding patients and ongoing. Sixteen patients (37%) required at least one dose reduction with a median time to first dose reduction of 2 months (0.5–9.6). The most common Grade 4 adverse event was neutropenia (14%) and the most common Grade 3 adverse events were neutropenia (21%) and thrombocytopenia (12%).

Conclusion: Lenalidomide oral monotherapy is active with manageable side effects in relapsed/refractory indolent NHL.
High Response Rate to Lenalidomide in Relapsed/Refractory Aggressive Non-Hodgkin’s Lymphoma with Prior Stem Cell Transplant.
Authors: Vose Julie M. , Tuscano Joseph M. , Justice Glen , Lossos Izidore S. , Ervin-Haynes Annette , Takeshita Kenichi , Pietronigro Dennis , Zeldis Jerome B. , Habermann Thomas M.
Published: April 2020

Background: High dose chemotherapy with stem cell transplant is currently employed in relapsed/refractory aggressive Non-Hodgkin’s Lymphoma (NHL). Lenalidomide (Revlimid®), an immunomodulatory drug, has shown activity in hematological malignancies including relapsed/refractory multiple myeloma, chronic lymphocytic leukemia and cutaneous T-cell lymphoma.

Aim: To determine the activity and safety of lenalidomide monotherapy in relapsed/refractory aggressive NHL following stem cell transplant (SCT).

Methods: Patients with relapsed/refractory aggressive NHL with measurable disease ≥ 2 cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology.

Results: Fourteen (29%) of the 49 patients enrolled into the study had a prior SCT. Median age was 61 (23–76) and 5 were female. Histology was diffuse large B-cell lymphoma [DLBCL] (n=5), follicular center lymphoma grade 3 [FL] (n=2), mantle cell lymphoma [MCL] (n=5) and transformed [TSF] (n=2). Median time from diagnosis to lenalidomide was 3.9 (1.1–31.4) years and median time from SCT to study entry was 1.9 (0.5–11.7) years. The median number of prior treatment regimens was 5 (2–8). Seven patients (50%) exhibited an objective response (1 complete response unconfirmed (CRu), and 6 partial responses (PR)), 5 had stable disease (SD) and 2 patients had progressive disease (PD). Six responses were in eight patients having a tumor burden < 50 cm2 and a time since last rituximab therapy of ≥ 230 days. One response was achieved in six patients having a tumor burden ≥ 50 cm2 or a time since last rituximab therapy of < 230 days. Four of 6 (67%) patients who had SCT as their last treatment prior to lenalidomide [median time from SCT to lenalidomide = 0.8 (0.5–4.8) years] responded. Progression free survival [PFS] is 4.5 months and ongoing. Six patients (43%) required at least one dose reduction with a median time to first dose reduction of 1.6 months (0.4–4.9). Two patients each (14%) had Grade 4 adverse events of neutropenia and thrombocytopenia. The most common Grade 3 adverse events were neutropenia (36%), thrombocytopenia (21%), and leukopenia (14%).

Conclusion: Lenalidomide produced a 50% response rate with manageable side effects in relapsed/refractory aggressive NHL following stem cell transplant.
Initial Results from an International Study in Relapsed/Refractory Aggressive Non-Hodgkin’s Lymphoma To Confirm the Activity, Safety and Criteria for Predicting Response to Lenalidomide Monotherapy.
Authors: Witzig Thomas E. , Reeder Craig B. , Polikoff Jonathan , Chowhan Naveed M. , Esseessee Isaac , Greenberg Richard , Patel Hermant , Vafai Davood , Wiernik Peter H. , Ervin-Haynes Annette , Pietronigro Dennis , Zeldis Jerome B. , Czuczman Myron
Published: April 2020

Background: Lenalidomide (Revlimid®) has demonstrated activity with manageable side effects in relapsed/refractory aggressive non-Hodgkin’s lymphoma (NHL). In the initial pilot study, response to lenalidomide was associated with low tumor burden, long duration from prior rituximab and high absolute lymphocyte count.

Aims:

To confirm the activity and safety of lenalidomide monotherapy in patients with relapsed/refractory aggressive NHL in an international setting and Determine whether predictors of response identified in the previous study were valid.

Methods: Patients with relapsed/refractory aggressive NHL with measurable disease ≥ 2 cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology. Univariate analyses using Fisher’s exact test were conducted to investigate and characterize associations of prognostic variables with response.

Results: Eighteen patients who received lenalidomide were eligible for response assessment as of May 15, 2007. The median age was 66 (43–81) and 17 were male. Histology was diffuse large B-cell lymphoma [DLBCL] (n=11), mantle cell lymphoma [MCL] (n=5) and transformed (n=2). Median time from diagnosis was 2 (0.4–12) years and median number of prior treatment regimens was 3 (1–13). Four patients (22%) exhibited an objective partial response including 2/11(18%) DLBCL and 2/5 (40%) MCL. Five patients had stable disease. Lenalidomide response was associated with low disease burden (29% for < 50 cm2v 0% for ≥ 50 cm2), time from last rituximab to lenalidomide treatment (36% for ≥ 230 days v 0% for < 230 days) and absolute lymphocyte count [ALC] (33% for ≥ 0.6×109/L v 0% < 0.6×109/L). Patients with favorable values for these three prognostic factors (N = 7) had a 57% response rate (compared with a 0% response rate for patients with unfavorable values (N = 11; P = 0.01). Grade 4 adverse events were neutropenia (22%) and thrombocytopenia (11%). Most common Grade 3 adverse events were fatigue (17%), thrombocytopenia (11%) and leukopenia (11%).

Conclusion: Lenalidomide has activity in relapsed/refractory aggressive NHL with a favorable safety profile. Initial results from this ongoing study are confirming that patients with low tumor burden, long duration from prior rituximab, and high ALC are more likely to respond.
Bortezomib, Rituximab, and Dexamethason (BORID) as Salvage Treatment in Relapsed/Refractory Mantle Cell Lymphoma: Sustained Disease Control in Patients Achieving a Complete Remission.
Authors: Drach Johannes , Kaufmann Hannes , Pichelmayer Oskar , Sagaster Verena , Holzer Sonja , Odelga Viktoria , Zielinski Christoph , Raderer Markus
Published: April 2020

Background: Bortezomib (B) belongs to a new class of anti-cancer agents, the proteasome inhibitors, and has documented activity in multiple myeloma and mantle cell lymphoma (MCL). Preclinical studies suggest that B has synergistic activity with rituximab (R), which provides a rationale for the exploration of treatment combinations. We therefore evaluated the activity and safety of B in combination with R and dexamethasone (BORID) in patients with relapsed and refractory MCL (phase II trial).

Methods: A treatment cycle consists of B at 1.3 mg/m2 administered on days 1, 4, 8, and 11, R at 375 mg/m2 administered on day 1, and dexamethasone 40 mg orally on days 1 to 4. Cycles are repeated every 3 weeks for a total of 6 treatment cycles. Patients (pts) achieving a response received 4 additional doses of R as maintenance (every 8 weeks). Pts with progressive MCL after at least one prior line of therapy (including CHOP or a CHOP-like regimen) were eligible.

Results: We have completed enrollment of 16 pts (median age, 67 years; range, 48 to 75 years) after a median of 3 lines of prior therapies (range, 1 to 6, prior rituximab in 88%; thalidomide in 50%; high-dose therapy in 31%; a fludarabine-containing regimen in 31%). Median time between start of frontline therapy and study inclusion was 42 months (range, 11 to 98 months). Severe adverse events (> grade II) included infections (herpes zoster in 2 pts, bacterial pneumonia, mucosal candidiasis), peripheral neuropathy (3 pts), fatigue (2 pts) and vasculitic skin infiltrates in 3 pts. Thrombopenia (< 50 G/L) occured in 2 pts. All adverse events were managable by standard means of supportive care and prolongation of the treatment interval between cycles. Overall response rate was 69% (11 of 16 pts), with 6 pts achieving a CR (38%; confirmed by PET-scan in 5 pts) and 5 pts reaching a PR. Skin infiltrates (histologically proven T-cell infiltrates) preceded achievement of CR in 2 pts. Remission status appeared to be associated with progression-free survival (PFS): Patients in CR had longer PFS (29+, 24+, 21+, 12+, 12, and 10+ months) compared to patients in PR (median 8.5 months, range 6 – 15).

Conclusions: BORID has promising activitiy (69% overall response rate; CR rate 38%) and managable toxicity in this patient population with predominantly heavily pretreated MCL. Achievement of a CR emerged as an important factor for sustained disease control. Further evaluation of this regimen, in particular in pts at an earlier phase of the disease, is warranted.
First Report of a Phase II Clinical Trial of Lenalidomide Oral Therapy for Peripheral T-Cell Lymphoma.
Authors: Reiman Tony , Finch Daygen , Chua Neil , White Darrell , Stewart Douglas A. , van der Jagt Richard , Johnston James , Prasad Angeli , Schwarz Henry , Zeldis Jerome , Belch Andrew R.
Published: April 2020

BACKGROUND: Lenalidomide has immunomodulatory and anti-neoplastic properties, with demonstrated activity in myelodysplastic syndrome and multiple myeloma. Preliminary reports indicate that lenalidomide has activity against cutaneous T-cell lymphoma (mycosis fungoides) and chronic lymphocytic leukemia. We hypothesized that lenalidomide should be studied as a non chemotherapy approach for Peripheral T- Cell Lymphoma (PTCL).

STUDY DESIGN AND METHODS: In this Canadian multi-center, open label, single-arm, phase II clinical trial, patients with PTCL were treated with lenalidomide 25 mg po qd × 21 days on a 28-day cycle until disease progression, death or unacceptable toxicity. Patients with ECOG 0-2 and relapsed/refractory disease were eligible, as well as patients who had not previously had systemic therapy but who were ineligible for standard curative chemotherapy regimens due to comorbid illness. We report on the first ten patients enrolled. The primary endpoint is response rate defined according to the 1999 Cheson criteria.

RESULTS: Median age of participants was 56y (range, 42–76y), 9M, 1F. Histologies included PTCL not otherwise specified (n=4), angioimmunoblastic (n=4), cutaneous anaplastic large cell (n=1), hepatosplenic gamma/delta (n=1). 8 were relapsed, 2 previously untreated. 2 were refractory to their previous regimen. Median number of prior lines of systemic therapy is 1 (range, 0–3). Median number of cycles delivered to date, 2 (range: 1–8). Therapy has generally been well tolerated. 3 patients experienced grade 3–4 hematological toxicity, 3 experienced grade 3+ infectious complications and 1 had grade 3 rash. No thrombotic events have been seen to date. Best responses to date include 0 CR, 4 PR (2 angioimmunoblastic, 2 PTCL NOS), 1 SD (PTCL NOS), 2 PD (1 angioimmunoblastic, 1 PTCL NOS). Three deaths have occurred on study, due to disease progression (n=2) and pneumonia during cycle 1 (n=1, angioimmunoblastic). One patient has withdrawn from study post cycle 1 due to treatment related asthenia. One previously untreated patient still awaits the first response assessment; the overall response rate (CR+PR) to date in the remaining patients on an intent-to-treat basis is 4/9 (44%). 5 of 9 (56%) patients have achieved stable disease or better, for 2+, 3+, 5, 6 and 8+ months. For the two patients with previously refractory disease, best response was 1 PR for 6 months and 1 PD.

CONCLUSION: Lenalidomide appears to be an active agent in the treatment of relapsed PTCL with an acceptable tolerability profile. Further recruitment and follow-up will allow us to better define the response rate, tolerability, TTP and OS with this regimen.
Phase I Trial of Fenretinide (4-HPR) Intravenous Emulsion for Hematologic Malignancies.
Authors: Mohrbacher Ann , Gutierrez Martin , Murgo Anthony J. , Kummar Shivaani , Reynolds C. Patrick , Maurer Barry J. , Groshen Susan , Vergara Lori , Kang Min H. , Yang Allen S.
Published: April 2020

Background: 4-HPR is a cytotoxic retinoid with broad anti-cancer activity in preclinical studies, but oral capsule 4-HPR had limited bioavailability and activity in clinical trials. An intravenous intralipid emulsion formulation of 4-HPR (ILE 4-HPR) was developed to increase 4-HPR systemic exposure.

Methods: ILE 4-HPR was administered as a continuous intravenous infusion for 5 of every 21 days and systemic toxicities, clinical response, and pharmacokinetics (PK) assessed. Simon design dose escalation proceeded with a 100% increase per dose level until moderate toxicity in 2 patients or 1 dose-limiting toxicity (DLT). Ten dose levels were planned starting at 80 mg/m2/day, increasing until 1810 mg/m2. Plasma 4-HPR levels were measured by high performance liquid chromatography.

Results: To date, 17 patients have been enrolled. At dose level 10 (1810 mg/m2/day), 2 patients experienced a DLT of grade 4 hypertriglyceridemia, 1 patient with transient grade 2 pancreatitis. A de-escalation to dose level 9 (1280 mg/m2/day) enrolled 5 patients: 1 had asymptomatic Grade 4 hypertriglyceridemia, 1 patient experienced pleural effusions that resolved after pleurocentesis. A de-escalation to dose level 8 (905 mg/m2/day) enrolled 5 pts: two patients experienced asymptomatic Grade 4 hypertriglyceridemias that resolved after stopping the infusion; enrollment is ongoing. PK showed a linear relationship of dose to plasma level, with steady-state 4-HPR levels of 25 μM (640 mg/m2, level 7); 54 μM (1280 mg/m2, level 9) and 62 μM (1810 mg/m2, level 10). Responses to date include a transient response in a non-Hodgkins lymphoma at 320 mg/m2, in two angioimmunoblastic T-cell lymphomas, an 8-month partial response at 1810 mg/m2/day and a 4+ month unconfirmed complete response at 905 mg/m2/day, and in a histone deacetylase inhibitor-refractory cutaneous T cell lymphoma, a 10+ month molecular complete response at 1280 mg/m2. All patients were heavily pretreated. The DLT of hypertriglyceridemia is likely related to the intralipid formulation vehicle accounting for 5/6 DLTs observed, all reversible.

Conclusions: ILE 4-HPR can be safely administered and obtained 4-HPR plasma levels 6 to 7 times higher than previously obtained by oral capsule 4-HPR. Durable clinical activity was observed in T cell lymphomas in the dose range 905–1810 mg/m2.

Supported in part by NCI U01CA62505 and the NCI RAID program.
Co-Delivery of Sleeping Beauty Transposons Encoding FVIII and Indoleamine 2,3-Dioxygenase Attenuate Inhibitor Formation Resulting in Long-Term FVIII Expression.
Authors: Liu Li , Fletcher Bradley S.
Published: April 2020

Gene therapy in adult hemophilia A mice is known to generate a robust immune response against the newly produced FVIII protein. This phenomenon has been extensively reported for both viral and non-viral vectors. T cell activation promotes the proliferation of B cells that produce antibodies against FVIII (inhibitors) leading to a loss of circulating FVIII protein and activity. Approaches to attenuate this immune response have included the use of cytotoxic chemotherapeutic agents (such as cyclophosphamide) or the use of antibodies or soluble fusion proteins that prevent T cell activation (such as the fusion protein between the cytotoxic T lymphocyte-associated antigen 4 and the immunoglobulin heavy chain, CTLA-4-Ig). Recently, the mechanism by which CTLA-4-Ig exerts its effect has been elucidated and is thought to involve tryptophan catabolism by upregulation of the enzyme indoleamine 2,3-dioxygenase (IDO). Catabolites of tryptophan degradation, such as kynurenine, have been shown to block T cell proliferation and promote T cell apoptosis. Here we report that hydrodynamic co-administration of Sleeping Beauty transposons encoding both FVIII and IDO are able to attenuate, but not fully block, inhibitor formation. With this approach, long term expression of FVIII at therapeutic levels (∼10% of normal) can be achieved in adult animals without the need for additional immune suppression. Only in animals receiving FVIII and IDO together can we detect FVIII protein by western blot at 24 weeks. Serum kynurenine levels are slightly elevated after gene delivery in animals receiving the IDO gene, but fall to within normal levels by 24 weeks. These results suggest that modulation of the levels of tryptophan catabolites in vivo can influence the formation of FVIII inhibitors.
Mesenchymal Stromal Cells Genetically Engineered To Overexpress Insulin-Like Growth Factor-1 Provide Paracrine Support for Cell-Based Erythropoietin Therapy of Chronic Renal Failure-Induced Anemia.
Authors: Kucic Terrence , Copland Ian B. , Cuerquis Jessica , Coutu Daniel L. , Chalifour Lorraine E. , Gagnon Raymonde F. , Galipeau Jacques
Published: April 2020

Mesenchymal stromal cells (MSC) are a population of non-hematopoietic progenitors native to the bone marrow that are amenable to genetic engineering, making them attractive delivery vehicles for the in vivo production of therapeutic proteins, such as erythropoietin (Epo). We have previously demonstrated that MSC engineered to secrete Epo can be used for the long-term correction of renal failure-induced anemia [Eliopoulos et al., J Am Soc Nephrol. June 2006]. However, limited long-term transplanted cell survival compromises the efficacy of MSC-based gene therapy approaches. The current study provides evidence that co-implantation of MSC overexpressing Insulin-like growth factor-1 (IGF-I) improves MSC-based gene therapy of anemia by providing paracrine support to Epo-secreting MSC within a synthetic subcutaneous organoid. The IGF-I receptor was found to be expressed in murine MSC by RT-PCR, and protein expression was confirmed by immunoblot. We also demonstrated MSC MAPK pathway responsiveness to IGF-I stimulation in vitro and subsequent improvement of MSC survival following staurosporin-induced apoptosis. Murine MSC were transduced to overexpress either Epo or IGF-I (hereafter MSC-Epo and MSC-IGF) using retroviral vectors. MSC-Epo were subsequently admixed in a collagen matrix and implanted by subcutaneous injection in both naïve mice and a murine model of chronic renal failure, in combination with either MSC-IGF or null MSC. Mice receiving MSC-Epo in conjunction with MSC-IGF experienced a greater and significantly sustained elevation in hematocrit compared to control mice. In addition, mice co-implanted with MSC-IGF and MSC-Epo demonstrated a significant improvement in cardiac function compared to controls. In conclusion, cell-based gene therapy using co-implanted MSC-IGF represents a promising new strategy for the treatment of renal failure-induced anemia, as well as for the improvement of gene-enhanced MSC survival within implanted matrices.
Intravenous Administration of Manganese Superoxide Dismutase-Plasmid Liposomes (MnSOD-PL) in a Mouse Model Protects Against Whole Body Irradiation.
Authors: Greenberger Joel S. , Smith Tracy , Schlesselman James J. , Epperly Michael W.
Published: April 2020

Organ specific localized administration of manganese superoxide dismutase plasmid/liposome complexes (MnSOD-PL) confers protection of the rodent lung, esophagus, oral cavity, intestine and bladder from single fraction and fractionated ionizing irradiation by a mechanism dependent upon mitochondrial localized antioxidant effects. Intravenous injection of MnSOD-PL significantly increased survival of both male and female C57BL/6NHsd mice from the LD 50/30 whole body irradiation dose of 9.5 Gy (p = 0.0001 or 0.0059, respectively). To determine if systemic MnSOD-PL mediated improved survival at the expense of a deleterious delayed increase in development of cancer, life shortening, or neurodegenerative disease we followed all survivors. Fifty male and female C57BL/6NHsd mice per group were injected intravenously with MnSOD-PL (100 μg plasmid DNA in 0.1 ml) and irradiated with control mice to 0, 1 or 9.5 Gy whole body irradiation 24 hrs later. Moribund or dying mice were sacrificed, examined for tumors and bone marrow isolated stained with Wright Geimsa, and examined for abnormal hemogram. Small intestine was fixed in formalin, sectioned and examined for intestinal changes. Female mice pretreated with MnSOD-PL prior to 9.5 Gy (LD 50/30) had an increased survival over the first 30 days which is the time frame for death due to hematopoietic or intestinal damage with 90% survival compared to 58% for the control irradiated mice (p = 0.0059). Between 30 and 530 days there was no significant change in survival rate between MnSOD-PL treated and control irradiated 9.5 Gy or 1.0 Gy groups. Beginning at day 400, all groups have a similar death rate which can be attributed of the age of the mice. Male mice pretreated with MnSOD-PL and irradiated to 9.5 Gy had 75% survival after 30 days compared to 25% for the control mice (p < 0.0001). Between 30 and 375 days after irradiation, there was no significant change in survival rate between MnSOD-PL treated compared to control irradiated 9.5 or 1.0 Gy groups. There was no detectable difference in the incidence of deaths associated with tumor (thymoma), marrow, or intestinal damage. All mice are being followed until they become moribund as a result of irradiation or old age. The data indicate, at this time, that systemic MnSOD-PL treatment is not detectably harmful to surviving total body irradiated mice.
High Frequency of Loss of Heterozygosity Due to Uniparental Disomy or Allele Deletion of Ocular Adnexal MALT-Type Lymphoma.
Authors: Asakura Yoshitaka , Ogawa Seishi , Kato Motohiro , Yamamoto Go , Maeshima Akiko , Matsuno Yoshihiro , Suzuki Shigenobu , Kaneko Akihiro , Nomoto Junko , Tanimoto Kazuki , Sekiguchi Naohiro , Maruyama Dai , Kim Sung-Won , Watanabe Takashi , Tobinai Kensei , Kobayashi Yukio
Published: April 2020

Majority of malignant lymphoma arising from the ocular adnexae are ocular adnexal MALT lymphomas (OAL). Several genetic abnormalities, including t(14;18)(q32;q21), trisomy 18 and trisomy 3 have been reported in OAL. However, none of them are found in more than half of cases with OAL by conventional methods. High density Single Nucleotide Polymorphism (SNP) array analysis with CNAG/AsCNAR algorithm allows high-resolution and genome-wide detection of both loss of heterozygosity (LOH) and copy number abnormality, especially uniparental disomy (UPD), without depending on the availability of paired normal DNA (Yamamoto et al, Am J Hum Genet. 2007; 81:114–26). UPD is acquired by somatic recombination and therefore not detected by conventional cytogenetic analysis or array CGH. In this study we analyzed DNA from OAL for the presence of LOH with or without copy number changes. Tissue samples from patients with OAL at our institute between 1995 and 2003 (N=32) were subject to SNP-array (250K NspI) analysis with CNAG/AsCNAR algorithm. The patients included 21 males and 11 females, and the median age of 56.5 years at the time of diagnosis (range, 15–90 years). Clinical stage was I (29 cases), II (1 case), and IV (2 cases). Trisomy of 3, 18, and 21 were found in 9, 7 and 1 cases, respectively. Overall, LOH due to UPD more than one chromosomal band were found in 16 cases (50%). Recurrent one allele deletion was detected at 6q23-24, 12p13 and 17q21 (N=15, 15 and 21, respectively). In total, 28 (82%) among 32 cases showed one or more of the above changes. Characterization of these recurrent genetic abnormalities might reveal the pathogenesis of OAL.
Cytokine-Dependent Imatinib Resistance in Mouse Bcr-Abl(+), Arf-Null Lymphoblastic Leukemia.
Authors: Williams Richard T. , Besten Willem den , Boulos Nidal , Sherr Charles J.
Published: April 2020

Retroviral vector-mediated transduction of the BCR-ABL tyrosine kinase into Arf-deficient mouse bone marrow progenitors allows rapid ex vivo outgrowth of pure pre-B cell populations that induce a highly aggressive form of ALL when inoculated intravenously into healthy syngeneic mice. These leukemias resist therapy with the BCR-ABL kinase inhibitor imatinib, and surprisingly, drug resistance is non-tumor-cell autonomous. We now demonstrate that expression of the p185 BCR-ABL isoform (hereafter p185) in Arf-null pre-B cells is sufficient to generate polyclonal populations of leukemia-initiating cells (LICs), only 20 of which induce fatal, imatinib-resistant disease within one month after infusion. These p185+ Arf-null LICs are pre-B cells by immunophenotypic criteria, lack evidence of myeloid and stem cells markers, exhibit immunoglobulin heavy chain gene rearrangements and random proviral insertions, and are almost universally capable of initiating lethal, transplantable leukemias in healthy syngeneic recipient mice. Therefore, these LICs do not represent rare ‘cancer stem cells’. Resistance to targeted therapy with imatinib in this model system is not dependent upon mutations within the BCR-ABL kinase domain, but, rather, is mediated through interactions between the LICs and the host environment. While cytokines like IL-7 are dispensable for the efficient generation of p185+, Arf-null cells, these LICs remain IL-7-responsive and are significantly protected from imatinib-induced cytostasis by IL-7 in vitro. Introduction of p185 into Arf-null hematopoietic progenitors that also lack the common gamma chain (γc) for cytokine receptors bypasses their cytokine requirements and permits pre-B cell development. These p185+, Arf-null, γc-null LICs also initiate ALL but exhibit much greater sensitivity to imatinib in vivo, allowing a significant fraction of treated mice to sustain long term remissions even when therapy is discontinued. Therefore, salutary cytokines expressed in the hematopoietic microenvironment facilitate leukemic proliferation and confer resistance to targeted therapy. Not surprisingly, single agent therapy with dasatinib, a second generation BCR-ABL kinase inhibitor, is dramatically more efficacious in this pre-clinical Ph+ ALL model, although some recipient mice still succumb to disease despite continuous drug treatment. Using vectors co-expressing p185 and luciferase, we are now initiating dasatinib therapy when recipient animals have detectable disease burdens, thereby permitting a more dynamic assessment of therapeutic response and subsequent failure. We speculate that in human Ph+ ALL, the frequent deletion of the INK4A-ARF locus (∼50% of cases at diagnosis) might help to maintain LIC survival in the face of targeted therapy and so facilitate the emergence of drug-resistant BCR-ABL variants. As such, INK4A-ARF deletion might prove to be a poor prognostic indicator.
The MMSET Gene Contributes to the Growth, Adhesion, and Tumorigencitiy of t(4;14) Multiple Myeloma Cells.
Authors: Lauring Josh , Abukhdeir Abde M. , Konishi Hiroyuki , Garay Joseph P. , Gustin John P. , Wang Qiuju , Arceci Robert J. , Matsui William , Park Ben H.
Published: April 2020

Multiple myeloma (MM) is an incurable hematological malignancy characterized by recurrent chromosomal translocations. The t(4;14)(p16;q32) is associated with the worst prognosis of any patient subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves two potential target genes on chromosome 4: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally over-expressed in t(4;14) MM, whereas FGFR3 expression is lost in one third of cases, suggesting a role for MMSET in myeloma pathogenesis. Nonetheless, the role of MMSET in t(4;14) MM has remained unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. Using homologous recombination-mediated gene targeting, we disrupted the N-terminal and full-length isoforms of MMSET in t(4;14)+ KMS-11 MM cells. Disruption of the translocated MMSET allele revealed that this allele accounts for most of the MMSET transcription in t(4;14) MM cells. Accordingly, selective targeting of the translocated allele, but not the non-translocated allele, led to reduced colony formation in methylcellulose and reduced tumor formation in nude mouse xenografts. Down-regulation of MMSET expression in t(4;14) MM cell lines by stable RNA interference (RNAi) led to a slower growth in liquid culture, a significant reduction in colony formation in methylcellulose, and decreased tumorigenicity in vivo. Additionally, MMSET knockdown led to partial cell cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Cells with targeted disruption or knockdown of MMSET exhibited changes in expression levels of potential target genes, including several adhesion molecules. These results provide the first direct evidence that translocation-mediated overexpression of MMSET plays a critical role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.
Constitutive Expression of Lef-1 in Murine Hematopoietic Progenitors Causes Myeloid and Lymphoid Leukemias Propagated by a Leukemic Stem Cell Population with Lymphoid Characteristics.
Authors: Petropoulos Konstantin , Schessl Christina , Arseni Natalia , Rawat Vijay P.S. , Deshpande Aniruddha , Stadler Christiane , Hiddemann Wolfgang , Quintanilla-Martinez Leticia , Bohlander Stefan K. , Feuring-Buske Michaela , Buske Christian
Published: April 2020

Canonical Wnt signaling is critically involved in normal hematopoietic development and the self-renewal process of hematopoietic stem cells. Deregulation of this pathway has been linked to a large variety of cancers including leukemia. Lef-1, a key factor of the Wnt / beta-catenin signaling pathway, plays pivotal roles in lymphoid development, but little is known about the role of Lef-1 in myeloid hematopoiesis and leukemogenesis. We now show that Lef-1 is expressed in murine hematopoietic stem cells (‘LSK’, Sca+cKit+Lin−) and both myeloid and lymphoid subpopulations as well as in different human leukemias. Using a retroviral BM transplantation model, we demonstrate that ectopic expression of wild type Lef-1 (WT) and a constitutive active mutant of Lef-1 (CA) induces a severe disturbance of normal hematopoietic development: mice transplanted with bone marrow constitutively expressing Lef-1 had a significant increase in the number of circulating myeloid cells resulting in an inverted lymphoid-myeloid ratio in the peripheral blood (ratio: 0.28 (WT), 0.10 (CA) vs. 1.07 (GFP); p<0.002). With a median latency of 12 month, transplanted mice succumbed to a lethal myeloproliferation (n=2) or acute myeloid or B-lymphoblastic leukemias (N=8). Both leukemia subtypes shared key biological characteristics such as positivity for IG DH-JH rearrangements. In addition, both subtypes were characterized by a biphenotypic cell population (B220+Mac1+Gr1+, BMG+++) as well as varying numbers of B220+Mac1−Gr1− (B+MG−) cells. In both leukemia subtypes single DJH–rearranged B+MG− cells had the highest seeding efficiency and were able to give rise to both BMG+++ and B−MG+ cells in vitro (mean seeding efficiency: B+MG−: 17.9%, vs. BMG+++: 4.7%, B−MG+: 4.0, p=0.005). Strikingly, the frequency of leukemia-initiating cells in AML was highest in the B+MG− population as determined by limit dilution transplantation assays (B+MG−: 1 in 433 vs. BMG+++/B−MG+: 1 in 8491 cells, p<0.001). Expression analysis of malignant blast cells from both lymphoid and myeloid leukemias also revealed striking commonalities with regard to the transcription profile. Blast cells from both AML and ALL diseased mice uniformly showed expression of myelo-specific genes like C/ebpα and c-fms and lymphoid specific genes E2A and Ebf-1, but lacked expression of the B-cell differentiation regulator Pax-5. These findings demonstrate that balanced expression of Lef-1 is crucial for early normal hematopoietic differentiation and that deregulation of this factor induces the development of DJH-rearranged acute myeloid and lymphoid leukemias which are propagated by a leukemic stem cell with lymphoid characteristics.
PML Protects HIPK2 and p300 from SCF-Mediated Ubiquitin-Dependent Degradation To Activate Transcription.
Authors: Shima Yutaka , Shima Takito , Chiba Tomoki , Irimura Tatsuro , Kitabayashi Issay
Published: April 2020

The Pml gene is the target of t(15;17) chromosome translocation in acute promyelocytic leukemia. PML protein is known to localize in discrete nuclear speckles, named PML nuclear bodies (NBs). In NBs, PML interacts with several transcription factors, such as p53 and AML1, and their co-activators, such as HIPK2 and p300. PML activates transcription of their target genes. PML is thought to stabilize transcription factor complex and function as a mediator in transcription activation, but little is known about the molecular mechanism by which PML activates transcription. To clarify the role of PML in transcription regulation, we purified the PML complex and identified a novel F-box protein (FBP), Skp1, and Cullin1 (Cul1) in the PML complex by LC/MS/MS analysis. FBPs form SCF ubiquitin ligase complexes with Skp1, Cul1 and ROC1 and mediate recognition of specific substrates for ubiquitination. We found that the FBP that we identified here also forms a SCF complex with Skp1, Cul1 and ROC1. To identify substrates for the SCF complex, we tested several proteins that could bind to PML, and found that the FBP promotes degradation of HIPK2 and p300. These degradations were inhibited in the presence of a proteasome inhibitor, MG132. The FBP stimulated ubiquitination of HIPK2. These results suggest that the SCF promotes degradation of these proteins by the ubiquitin-proteasome pathway. The fact that the SCF is a part of the PML complex suggests that PML plays a role in the SCF-mediated degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. In order to clarify the role of PML in degradation of HIPK2 and p300, we tested effects of PML on the degradation and found that PML inhibited the SCF-mediated degradation of HIPK2 and p300 without inhibition of ubiquitination. To clarify roles of HIPK2, PML IV and the FBP in p53-dependent transcription, we performed reporter analysis using the MDM2 promoter in H1299 cells. Since the FBP promotes degradation of HIPK2, we initially thought that the FBP might inhibit activation of p53-dependent transcription by HIPK2 and PML IV. However, the FBP, HIPK2 and PML synergistically stimulated the p53-dependent transcriptional activation. Taken together our data suggest that the SCF-induced ubiquitination of transcription co-activators HIPK2 and p300 plays a critical role in transcriptional regulation, and that PML stimulates transcription by protecting HIPK2 and p300 from ubiquitin-dependent degradation.