bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure-Based Drug Design of c-Kit Inhibitors for Use in the Treatment of Acute Myeloid Leukemia.
Authors: Maxwell David S. , Pal Ashutosh , Peng Zhenghong , Shavrin Alexandr , Faderl Stefan , Harris David , Van Quin , Liu Zhiming , Verstovsek Srdan , Manshouri Taghi , Ferrajoli Alessandra , Kantarjian Hagop M. , Estrov Zeev , Bornmann William G.
Published: October 2019

Inhibitors of c-Kit kinase have shown clinical relevance in various myeloid disorders, including acute myeloid leukemia (AML). Research in our lab has been oriented towards structure-based drug design of c-Kit inhibitors based on the available crystal structure. We describe the design, synthesis, and preliminary results from the in-vitro testing of several c-Kit kinase inhibitors in both enzymatic and cell-based assays. The design resulted from in-silico screening of several targeted libraries via docking to the crystal structure of c-Kit, followed by aggressive post-filtering by several criteria to significantly bias synthesis efforts towards candidate compounds with best chance for success. This led to 128 structures built from 8 common structural cores, from which 2 cores were initially selected based on the synthetic feasibility. Five compounds were initially synthesized, and were immediately followed by 60 compounds with variations to probe local structure-activity relationships. The initial set of compounds, designated APCKxxx, was tested in a c-Kit kinase assay; two compounds were found to have an IC50 in the high nM to low uM range. These compounds have been tested in a MTT-based assay using OCIM2 and OCI/AML3 cell lines. In the c-Kit expressing OCI/AML3 cell line, all five compounds possessed an EC50 < 500 nM and two had and EC50 ~100 nM. Our most recent results show that these compounds also show efficacy in some imatinib-resistant cell lines. We will discuss these results and our strategies for the second generation of compounds that are optimized for better activity, selectivity, and ADME properties.
Expression of Glucocorticoid-Induced TNF Receptor Ligand on Acute Myeloid Leukemia Cells Mediates the Release of Immunosuppressive Cytokines and Impairs NK Cell-Mediated Immune Surveillance.
Authors: Krusch Matthias , Baltz Katrin M. , Baessler Tina , Kloss Mercedes , Kumbier Ingrid , Peterfi Andrea , Kanz Lothar , Salih Helmut R.
Published: October 2019

NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance of hematological malignancies including acute myeloid leukemia (AML). Among the molecules influencing host-tumor interaction are many members of the TNF superfamily, which mediate multiple cellular functions including cellular proliferation, differentiation and cell death. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and nuclear factor kappa B and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while solid tumors express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell cytotoxicity and IFN-γ production. Here we analyzed the role of GITR and its ligand in AML. We report for the first time that GITRL is expressed on primary AML cells in 18 of 30 patients as determined by FACS and RT-PCR analysis. Reverse signaling through GITRL using a recombinant GITR-Ig fusion protein induces the release of the immunoregulatory cytokines IL-10 and TNF as determined by ELISA. GITRL-mediated cytokine production of AML cells is abrogated by inhibition of mitogen activated protein kinase (MAPK) pathways as demonstrated by addition of the specific p38 MAPK inhibitor SB202190, the specific JNK inhibitor SP600125 and the specific ERK Inhibitor II. Furthermore, binding of AML-expressed GITRL to GITR on NK cells downregulates cellular cytotoxicity and IFN-γ production in AML-NK cell cocultures, which can be overcome by addition of GITR-blocking antibodies as determined by cytotoxicity assays and ELISA. Thus, our data indicate that GITRL expression in AML substantially influences tumor immunoediting and enables the escape of leukemia cells from NK cell-mediated immunosurveillance.
A Phase I Study of the Farnesyltransferase Inhibitor Tipifarnib in a Week- on Week-Off Dose Schedule in Acute Myelogenous Leukemia.
Authors: Kirschbaum Mark , Selwyn Stein Anthony , Tuscano Joseph , Zain Jasmine M. , Popplewell Leslie , O’Donnell Margaret R. , Karanes Chatchada , Krishnan Amrita , Wright John J. , Pulone Bernadette , Rincon Amalia , Frankel Paul , Forman Stephen J. , Edward Newman
Published: October 2019

Tipifarnib (R115777, Zarnestra®) is a methylquinolone analogue that is a potent inhibitor of farnesylation. The initial phase I study in leukemia by Karp and Lancet (Blood, 2001) showed clinical responses in 10/34 evaluable patients with AML on a 21 day schedule, with 600 mg bid identified as the MTD. Preclinical work shows that farnesyltransferase remains inhibited for seven days after tipifarnib, suggesting an alternate week dosing schedule. We report the results of a tipifarnib phase I dose escalation trial in AML on a week on week off schedule. Dose levels were 400, 600, 800, 1200, 1400, and 1600 mg BID on a standard 3+3 design. Eligibility was defined as relapsed or refractory AML after one to three prior induction regimens. A total of 30 patients have been accrued. with 27 patients evaluable for toxicity. Median age is 64.5 (range 33–75). Grade 3 toxicities were seen at dose levels 1 (400 mg bid)- metabolic acidosis and hepatic failure in a patient with progressive leukemia who had previous hepatic toxicity during chemotherapy, and level 5 (1200 mg bid)- grade 3 creatinine elevation. Other grade 1 and 2 toxicities included fatigue, nausea, anorexia, elevated liver enzymes, increased bilirubin, and renal insufficiency. The maximum target treatment dose, 1600 mg PO bid was attained. There were 3 complete responses (CR) out of 9 patients treated at the 1000–1200 mg bid dose level- after cycle 1 in a 47 year old woman with relapsed AML after autologous transplant, with a 10 month continuing remission as of 8/06 (she underwent successful allogeneic transplant); after 2 cycles in a 69 year old man with relapsed AML, who relapsed after 5 months, (with response after retreatment with tipifarnib), and in a 67 year old man with relapsed AML who achieved PR after one cycle and CR after five cycles. One patient with AML evolving out of CMMoL had disappearance of the AML blasts but persistence of the CMMoL after two cycles at the 800 mg bid level, and one patient at the 1400 dose had a platelet rise to 140 from a starting platelet count below 40 despite continued presence of blasts.

In AML patients, greater than two fold increase in tipifarnib dosing can be tolerated on this dosing schedule with efficacy perhaps enhanced. Based on these promising results a monotherapy phase 2 or tipifarnib combination study in AML appears warranted.
Phase II Study of Clofarabine and Cytosine Arabinoside in Adult Patients with Relapsed AML and in Elderly Patients with Untreated AML Who Are at High Risk of Anthracycline Toxicity.
Authors: Agura Edward , Brian Berryman R. , Brougher Laura , Cooper Barry , De Leon Andrew , Fay Joseph , Holmes Houston , Maisel Christopher , Molina Arturo , Pineiro Luis , Saracino Giovanna , Tadic-Ovcina Mirjana , Vance Estil
Published: October 2019

PURPOSE: To evaluate the efficacy and safety of clofarabine, a novel deoxyadenosine analog, in adult patients with refractory or relapsed acute myeloid leukemia and in selected elderly patients with untreated AML and heart disease.

PATIENTS AND METHODS: In a phase II, open-label trial, 23 patients with de-novo AML, AML evolved from MDS or relapsed AML received a 5 day regimen consisting of clofarabine 40 mg/m2 intravenously over 1 hour followed within 4 hours by Ara-C 1000 mg/m2. The median age was 68 years (range 39–79 years). Fifteen (65%) had received at least one prior cytotoxic regimen (excluding 5-AZA). Significant cardiovascular disease (history of myocardial infarction, bypass grafting, cardiomyopathy) was present in 52% (12/23) prior to therapy.

RESULTS: 22/23 received at least one cycle of therapy and 5 received 2 cycles. One early death was due to disease progression. Grade 4 neutropenia developed in all patients. There were no cases of regimen-related cardiac toxicity. Most patients had some degree of edema and third-spacing syndrome. Several developed a significant but reversible acral rash. 20 patients are evaluable for response. The histologic response rate (defined as marrow blasts <5%) is 60% (12/20) consisting of 10 (50%) complete remissions and 2 (10%) partial responses. Complete cytogenetic remissions occurred in 9 patients. Durable remissions and low toxicity allowed some patients to proceed to nonablative allogeneic stem cell transplantation.

CONCLUSION: Clofarabine (40mg/m2) and Ara-C (1000mg/m2) x 5 days is an active and well tolerated regimen in myeloid malignancies including “elderly AML”—a distinct entity usually associated with poor response rate and high treatment-related toxicities. Other drug combinations with clofarabine are being explored for use in allogeneic transplant regimens and with other high-risk patient groups.
Phase I Study of XL-119, a Rebeccamycin Analog, in Patients with Refractory Hematological Malignancies.
Authors: Giles Francis J. , Ravandi Farhad , Cortes Jorge , Estrov Zeev , Faderl Stefan , Ferrajoli Alessandra , Garcia-Manero Guillermo , Thomas Deborah , Wierda William , Verstovsek Srdan
Published: October 2019

XL-119, a water soluble analog of the antibiotic agent rebeccamycin, has demonstrated broad spectrum antitumor activity in preclinical studies, including P388 and L1210 leukemia in murine models. XL-119 intercalates DNA, and inhibits topo II activity by a unique highly potent mechanism which renders it active in etoposide-resistant A549 and HCT 116 leukemia cell lines. On a Phase I study in patients with refractory hematological malignancies XL 119 was given as a 60 minute IV infusion, on D1–5 of 21 day cycles. The starting dose for the initial cohort was 140 mg/m2/d with planned escalation by 20% increments in the setting of a standard 3+3 design. A total of 28 patients (pts) have been treated to date. Three, 5, 3, 6, 3, and 8 have been enrolled to dose levels (DL) of 140, 160, 180, 200, 220 and 240 mg/m2/d, respectively. Median age was 57 years (range 19–82). Median ECOG status 1 (range, 0–2). Diagnoses included: AML (22 pts), MDS (1 pt) and ALL (5pts). AML was either primary refractory or at an advanced relapse state and had failed a median of 3 prior salvage treatments (range 1–10). The pt with MDS had received 4 prior therapies. Pts with ALL had received a median of 3 prior therapies. Dose limiting toxicities (DLTs) were observed in evaluable patients at the following dose levels: 1 patient at 200 mg/m2 experienced DLTs: grade 3 hand-foot syndrome and grade 4 mucositis. An expansion of this dose level yielded no further incidences of DLT. 1 patient at 240mg/m2 experienced DLT with grade 3 mucositis. The cohort was expanded without observation of additional DLTs. Dose level 260 mg/m2 is currently being explored with 2 patients too early for full evaluation. Two patients with refractory AML achieved a significant elimination of marrow blasts and received a second cycle of therapy. Twenty-four of 28 patients had transient reductions in peripheral blood blasts. XL 119 is a well-tolerated myelosuppressive agent which warrants further study in patients with hematological malignancies.
Everolimus Is Comparably Effective to Rapamycin Acting Synergistically with Imatinib in the Treatment of Constitutively Active Mutations of the Juxtamembrane Domain of c-Kit.
Authors: Corbacioglu Selim , Prill Jan-Michael , Wagner Tobias , Debatin Klaus-Michael
Published: October 2019

Kit cITD is a complex internal tandem duplication involving the juxtamembrane (JMD) and kinase domain of stem cell factor receptor c-kit which was identified recently in 7% of childhood AML. kit cITD induces constitutive proliferation and apoptosis resistance that can be synergistically blocked with imatinib (im) and rapamycin (ra).

Due to a long half-life ra has disadvantageous pharmacological characteristics making drug handling especially difficult in children with serum concentrations quickly raising to toxic levels. We therefore compared ra with its ester analogue everolimus (RAD001, ev) which has a better bioavailability and a shorter half-life in order to assess equivalence and potential functional superiority. Ba/F3 cells, stably transfected with c-kit WT and kit cITD, were treated with im, ra and ev alone and in combination. In proliferation assays of WT and cITD cell lines ev was a significantly more potent inhibitor than ra. When ra or ev were combined with low im concentrations proliferation was synergistically inhibited with both drugs but again significantly better with ev. In cITD expressing cells ra and ev alone were comparably poor inducers of apoptosis. In assays combining im with ra or ev both combinations showed a significant and comparable synergism with an additional dose effect of the mTOR inhibitors between 3nM and 0.3 nM. In a NOD/SCID animal model all animals injected with kit cITD succumbed due to multi-organ infiltration of c-kit positive cells within 20 days. Survival was prolonged in animals treated with im/ra and ev. We therefore conclude that ev is at least equivalent to ra and can replace ra exploring a molecular based therapeutic attempt in susceptible children with identified mutations of the JMD of c-kit.
Long-Term Safety and Efficacy of Stem Cell Gene Therapy for ADA-SCID.
Authors: Aiuti Alessandro , Benninghoff Ulrike , Cassani Barbara , Cattaneo Federica , Callegaro Luciano , Andolfi Grazia , Mirolo Massimiliano , Scaramuzza Samantha , Marktel Sarah , Tabucchi Antonella , Carlucci Filippo , Eibl Martha , Aker Memet , Slavin Shimon , Ciceri Fabio , Miniero Roberto , Bordignon Claudio , Roncarolo Maria Grazia
Published: October 2019

Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal congenital disorder of the immune system associated with systemic toxicity due to accumulation of purine metabolites. We previously showed that retroviral-mediated ADA gene transfer into autologous hematopoietic stem/progenitor cells (HSC) allowed restoration of immune and metabolic functions. We have now enrolled eight ADA-SCID children (age: 7–67 months) in our phase I/II gene therapy trial in which HSC are combined with low intensity conditioning with busulfan (total dose 4 mg/Kg i.v.). Previous treatment included haploidentical bone marrow transplant (n=3) or long-term (>1 year) enzyme replacement therapy (PEG-ADA) (n=4) associated with insufficient immune reconstitution or severe autoimmunity. In the latter case, PEG-ADA was discontinued to favour the growth advantage for gene corrected cells. The patients received a median dose of 8.8x106/Kg bone marrow CD34+ cells (range 0.9–10.8), containing on average 26.2±9.6% transduced CFU-C. Five patients experienced ANC <0.5x109/L, which was extended beyond day +30 in two patients. With a median follow up of 3.1 years (range 0.4–5.9), no adverse events related to gene transfer have been observed. Long-term engraftment of transduced HSC was demonstrated by stable multilineage marking, persisting more than 5 years from gene therapy. The average proportion of transduced cells in the peripheral blood at one year post-gene therapy (n=6) was 5% for granulocytes, 95% for T cells, 56% for B cells and 62% for NK cells. Comparison of the insertion sites retrieved ex vivo from patients with those identified in pre-transplant transduced CD34+ cells showed no skewing in the profile of genome distributions or in the gene families hit by the vector, and no clonal expansion. In the six children with a follow-up >1 year after gene therapy, we observed a progressive increase in lymphocyte counts which was sustained over time (median at 1.5 years 1.6x109/L), polyclonal thymopoiesis and normalization of T-cell functions in vitro. Serum Ig levels improved and evidence of antigen-specific antibodies was obtained, leading to IVIG discontinuation in five patients. All the children are currently healthy and thriving, and none of them showed severe infections. Sustained ADA activity in lymphocytes and RBC resulted in a dramatic reduction of RBC purine toxic metabolites (dAXP<30 nmoles/ml in 5 patients) and amelioration of children’s growth and development. In summary, these data confirm that gene therapy is safe and efficacious in correcting both the immune and metabolic defect in ADA-SCID, with proven clinical benefit.
Prevalence and Prognostic Significance of FLT3 Mutations in Acute Myeloid Leukemia: Association of ITDs with Poor Outcome in Patients with Normal Cytogenetics.
Authors: Palmisano Michela , Ottaviani Emanuela , Grafone Tiziana , Testoni Nicoletta , Paolini Stefania , Rondoni Michela , Baccarani Michele , Martinelli Giovanni
Published: October 2019

Acute Myeloid Leukemia (AML) is a difficult disease to treat, and better treatments are needed. Molecular targeted therapy represents a novel therapeutic approach.

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately one third of patients with de novo AML and have been implicated in its pathogenesis. The leukemic blasts of most AML patients have the internal tandem duplications (ITDs) in the juxtamembrane region or point mutations in Asp835 and Iso836 codons in the activation loop of the kinase domain (TKD) of the FLT3 receptor. Both mutations result in constitutive FLT3 receptor activity and may play a significant role in leukemogenesis.

In this study we have analyzed the incidence and type of FLT3 mutations in a large series of newly diagnosed AML patients. Furthermore, we have evaluated the prognostic impact of FLT3 mutations.

The FLT3/ITD was determined by polymerase chain reaction (PCR). The mutations of D835 and I836 codons were determined by PCR followed by restriction enzyme digestion (PCR-RFLP). For the estimation of the statistic significance of the differences in the clinical-biological characteristics, between the mutated patients and wild-type patients, it has been used the Student’s test t for independent data. The probabilities of overall survival (OS) and disease free survival (DFS) were analysed by Kaplan-Meier method; the differences of OS and DFS, between the mutated patients and wild-type patients, were assessed using the log-rank test.

Both FLT3/ITD and FLT3/TKD mutations were found in 15%. Dual mutations were found in 2% of 126 patients. Among the FAB subtypes of AML, the rate of FLT3 aberrations was higher in M4 (27%) and M5 (26%). FLT3/ITD was associated to leukocytosis (106.8x10(e)9/l vs 30x10(e)9/l in FLT3-wt, p=0.015) and high percentage of circulating blast cells (82% vs 42% in FLT3-wt, p<0.0001). Differently, FLT3/TKD mutations were not associated with high white blood cells count and blast cells percentage.

FLT3 mutations were more prevalent in patients with normal karyotype (51%). In this group, DFS and OS were significantly inferior for patients with FLT3/ITD than patients withouth mutations (0 vs 5, p=0.0032; 5 vs 9, p=0.049, respectively). We have identified the FLT3/ITD as an independent poor prognostic factor in AML patients with normal cytogenetics. Therefore, targeting FLT3 mutations represents a potential therapeutic target for AML. These results suggest that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients with “standard risk” profile.
Involvement of the NOTCH1 and NOTCH2 Genes in B-Cell Lymphomagenesis.
Authors: Wlodarska Iwona , De Keersmaecker Kim , Cools Jan , De Wolf-Peeters Chris , Bosly Andre , Delos Monique , Michaux Lucienne , Vandenberghe Peter , Hagemeijer Anne , Marynen Peter
Published: October 2019

The NOTCH pathway is a key regulator of developmental choices, differentiation and function throughout the hematopoietic system. An aberrant activation of this pathway may underlie leukemogenesis, as shown in T-ALL cases with the rare t(7;9)(q35;q34) targeting NOTCH1 or with mutation(s) of NOTCH1 found in approximately 50% of T-ALL cases. We report here evidence for NOTCH1 and NOTCH2 rearrangements in B-cell lymphoma. Involvement of NOTCH1 was identified by the presence of dic(9;14)(q34;q32) and trisomy 12 in a case of chronic lymphocytic leukemia (CLL) at the time of transformation, and by st(9;22)(q34;q11) accompanied by t(14;18)(q32;q21)/IGH-BCL2 and t(8;14)(q24;q32)/IGH-CMYC in a follicular lymphoma (FL). FISH analysis of dic(9;14) and t(9;22) demonstrated the respective involvement of the IGH and IGL loci, and mapped both 9q34 breakpoints to the 5′ end of NOTCH1. Western blot analysis with cleaved Notch1 (Val1744) antibody was performed in both cases and hyperactive NOTCH1 signaling was found in the first case only. This particular case displayed in addition a mutation in the PEST domain of NOTCH1. No mutations, neither in the PEST, nor in HD domain of NOTCH1 (2 domains frequently mutated in T-ALL) were found in the FL case. FISH analysis of 30 additional lymphoma cases documented by structural aberrations of 9q33q34 revealed a normal status of NOTCH1.

NOTCH2 rearrangement was identified by FISH in 1 out of the 3 leukemia/lymphoma cases we could collect with t(1;14)(p13;q32). This particular CLL case showed a 3-way translocation, t(1;14;18)(p13;q32;q21), targeting also BCL2. So far, this patient did not show any clinical features of CLL transformation.

We hypothesize that IG-translocations affecting NOTCH genes in B-cell malignancies contribute to the lymphomagenesis by deregulating the transcription of these genes resulting in an aberrant ligand-independent NOTCH signaling. Mutation in the PEST domain of NOTCH1 as found in one case with dic(9;14) may further contribute to the process by stabilization of the aberrant product. Further molecular and immunohistochemical investigations of these cases are in progress.
Inhibition of Polo-Like Kinase 3 by CIB1 Facilitates Outside-In Signaling through αIIbβ3 in Platelets.
Authors: Naik Ulhas P. , Naik Meghna U.
Published: October 2019

Integrin αIIbβ3, the platelet fibrinogen receptor, is involved in bi-directional signaling during platelet activation. It has been shown that the β3 subunit of this receptor is phosphorylated on both threonine and tyrosine. Although, the role of tyrosine phosphorylation is well studied, the role of threonine phosphorylation is not well understood. Here we identify the kinase responsible for threonine phosphoruylation of β3 and provide a mechanistic significance of this phosphorylation in outside-in signaling. We found that calcium- and integrin-binding protein 1 (CIB1), which specifically interacts with the cytoplasmic domain of αIIb, is not involved in inside-out signaling, but regulates outside-in signaling through αIIbβ3. CIB1 is known to interact with and regulate several serine/threonine protein kinases. Among these, p21 activated kinase 1 (PAK-1) is highly expressed in platelets. We found that CIB1 does not colocalize with PAK-1 during outside in signaling induced by adhesion of platelets to immobilized fibrinogen. When searched for the expression of other kinases that are known to interact with CIB1, we found that polo-like kinase 3 (Plk3) is expressed in platelets and is responsible for the constitutive phosphorylation of β3. Plk3 is constitutively active in resting platelets but is rendered inactive during outside in signaling as a result of CIB1 binding in a calcium dependent manner. Plk3 becomes colocalized with CIB1 at the filopodia when platelets change shape in response to an agonist and remain associated with both CIB1 and αIIb as platelets spread on immobilized fibrinogen, as indicated by immunoprecipitation experiments. These results suggest that in resting platelets, constitutively active Plk3 maintains β3 in a threonine phosphorylated state which is nonpermissive for activation. Upon platelet activation by agonist CIB1 associates with Plk3 and renders it inactive in a calcium-dependent manner. This allows serine/threonine phosphatases, such as PP1-c to dephosphorylate β3 on threonine and facilitate outside-in signaling. Thus we have identified the kinase responsible for threonine phosphorylation of β3 and have provided a possible mechanism of the regulation of outside-in signaling.
Congestive Heart Failure Is a Rare Event in Patients (pts) Receiving Imatinib Therapy.
Authors: Atallah Ehab , Durand Jean-Bernard , Kantarjian Hagop , Jabbour Elias , O’Brien Susan , Rios Mary , Cortes Jorge
Published: October 2019

Background: A recent report suggested that imatinib is cardiotoxic and can lead to severe left ventricular dysfunction and heart failure.

Study Aim: To evaluate the incidence of congestive heart failure (CHF) in pts receiving imatinib.

Methods: We reviewed all Grade III and IV cardiac or what could be considered cardiac-related adverse events (eg, shortness of breath, dyspnea on exertion, edema, etc) occurring in pts on clinical trials at MDACC involving imatinib.

Results: 1276 pts were enrolled on clinical trials with imatinib from 7/28/1998 to 7/27/2006. After reviewing all reported adverse events, particularly those that could be considered of at least possible cardiac origin, 22 pts (1.8%) were identified as having symptoms that could be attributed to CHF. The median age for these 22 pts was 70 yrs (range, 49 to 83 yrs); 12 were male. Their diagnosis at the time of imatinib was started was chronic myeloid leukemia in chronic phase (11 of 561 pts, 1.96%), accelerated phase (4 of 384 pts, 1.04%), or blast phase (2 of 123 pt, 1.62%), myeloproliferative disorder (4 of 124 pts, 3.2%), acute lymphoblastic leukemia (1 of 74 pts, 1.35%) and none in 10 pts with c-kit positive AML. Twelve pts (55%) had received prior interferon therapy and 3 pts had received anthracyclins. The median time from start of imatinib therapy to a cardiac adverse event was 162 days (2–2045 days). Imatinib dose at the time of AE was 300mg/d in 1 pt, 400mg/d in 8 pts, 600mg/d in 9 pts, and 800mg/d in 5 pts. At the time these events were reported, 8 were considered possibly or probably related to imatinib. Eighteen pts had previous medical conditions predisposing to cardiac disease: CHF (6 pts, 27%), DM (6 pts, 27%), hypertension (10 pts, 45%), coronary artery disease (CAD) (8 pts, 36%), arrhythmia (3 pts, 14%) and cardiomyopathy (1 pt 5%). Fifteen pts had an echocardiogram or MUGA scan at the time of the event, and 9 of them had documented low ejection fractions (<50%); in only 2 of these 9 pts was there an echocardiogram prior to imatinib (LVEF >50% in both). Of the 9 pts with low ejection fractions at the time of the event , 6 had prior cardiac conditions (3 CAD, 2 CHF and 1 cardiomyopathy), one patient’s low EF etiology could not be determined as no further cardiac evaluation was performed because of progressive disease, and one pt was on anagrelide prior to the event. Eleven of the 22 pts continued imatinib therapy with dose adjustments and management for the CHF symptoms with no further complications.

Conclusions: Imatinib therapy as a causal factor of CHF is uncommon and is mainly seen in elderly patients with pre-existing cardiac conditions. Patients with a previous cardiac history should be monitored closely and treated aggressively with diuretics if they develop fluid retention.
In Vitro and In Vivo Activity of SKI-606, a Novel Abl/Src Inhibitor, Against Imatinib Resistant BCR-ABL+ Neoplastic Cells.
Authors: Puttini Miriam , Coluccia Addolorata M.L. , Boschelli Frank , Franceschino Anna , Tornaghi Lucia , Ahmed Shaheen , Redaelli Sara , Piazza Rocco , Magistroni Vera , Andreoni Federica , Scapozza Leonardo , Formelli Franca , Gambacorti Carlo
Published: October 2019

Resistance to imatinib represents an important scientific and clinical issue in CML and Ph+ ALL and is usually due to the selection of cells harbouring kinase point mutations of the BCR-ABL gene or presenting BCR-ABL gene amplification. In order to improve Bcr-Abl inhibition, several inhibitors with higher potency compared to imatinib have been identified including dasatinib and nilotinib, which show clinical activity in patients resistant to imatinib. In the present study the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 is a selective dual Src/Abl inhibitor potently active against several CML cell lines and transfectants with IC50 values in the low nanomolar range, 2 logs lower than those obtained with imatinib. Cells expressing activated forms of c-KIT (HMC1560 and GIST882) or PDGFR (Ba/F3 Tel-PDGFRβ) were unaffected by SKI-606, in contrast to imatinib and dasatinib. SKI-606 reduced proliferation rate of cells where resistance to imatinib was caused by BCR-ABL gene amplification, such as Lama84R and of imatinib-resistant K562R and KCL22R in which no known mechanism have been identified. D276G, Y253F and E255K kinase point mutants were affected by SKI-606, resulting in inhibition of cell proliferation with IC50 values of 25, 40 and 394 nM (imatinib:1147, 1888 and 3174 nM). No activity was observed against T315I mutant. These results were confirmed in in vivo experiments, in models where resistance was not caused by mutations, as well as in cells carrying the Y253F, E255K and D276G mutations. In the first one SKI-606 was able to eradicate tumors, while significant delay in tumor growth was observed using Ba/F3 transfectants. Modelling considerations attribute the superior activity of SKI-606 to its ability to bind the intermediate (1M52) conformation of Bcr-Abl, in contrast to imatinib.

Excluding the T315 residue involved in direct hydrophilic interactions with both SKI-606 and imatinib, Y253 and E255 make electrostatic interactions stabilizing the inactive conformation of Abl nucleotide binding loop, thus affecting in a more pronounced way the binding of imatinib. Similar considerations have been made for the D276 residue affecting a-Helix C inactive conformation. The extended profile of activity performed here allows us to conclude that SKI-606 is a fairly active and specific inhibitor, with a very limited number of targets outside the Abl and Src families of TK.
Orally Active Inhibitors of the Imatinib Resistant Bcr-Abl Mutant T315I.
Authors: Shakespeare William C. , Wang Frank , Xu Qihong , Zhu Xiaotian , Narasimham Narayana , Metcalf Chester A. , Wang Yihan , Sundaramoorthi Raji , Huang Wei-Sheng , Zou Dong , Thomas Mathew , Romero Jan , Wen David , Chen Ingrid , Cai Lisi , Liu Shuangying , Wardwell Scott , Ning Yaoyu , Keats Jeff , Snodgrass Joseph , Russian Karin , Broudy Marc , O’Hare Thomas , Smith Caitlyn , Corbin Amie , Druker Brian , Iuliucci John , Dalgarno David , Sawyer Tomi K. , Clackson Tim
Published: October 2019

Resistance to the Bcr-Abl kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML) is associated with emergence of Bcr-Abl point mutations that preclude effective drug binding. Although most mutants are effectively inhibited with the second generation inhibitors dasatinib and nilotinib, neither compound inhibits the T315I mutant which represents ~ 25% of all clinically observed mutants. Through our program of structure-guided design, we have identified a series of compounds that inhibit the T315I mutant of Bcr-Abl both in vitro and in vivo. AP24534, a representative member of this new series, inhibited the kinase activity of both the wild-type enzyme and the T315I point mutant with IC50s of 3 and 31 nM respectively, and inhibited the proliferation of their respective BaF3-derived cell lines with IC50s of 2 and 14 nM. Additionally, AP24534 inhibited the proliferation of BaF3 cells expressing the clinically relevant mutants Y253F, E255K, H396P, or M351T with IC50s of 2, 7, 1, and 1 nM respectively. Inhibition of cellular proliferation directly correlated with decreased cellular phosphorylation of Bcr-Abl. Daily oral administration of AP24534 to mice bearing subcutaneous xenografts of Bcr-Abl-T315I-expressing BaF3 cells elicited dose-dependent tumor shrinkage, with complete tumor regression observed at the highest doses. In a separate model, daily oral administration of AP24534 significantly prolonged the survival of mice injected intravenously with these cells, at doses ranging from 5–30 mg/kg. These data indicate that this class of inhibitors has the potential to address CML refractory to current targeted agents.
Epigenetic Dysregulation of Candidate Cis-Regulatory Sequences in Hematological Malignancies.
Authors: Thompson Reid F. , Figueroa Maria E. , Melnick Ari M. , Greally John M.
Published: October 2019

Epigenetic changes (in particular, altered cytosine methylation) have been described in a variety of tumors. The CpG Island Methylator Phenotype (CIMP) is a well-known instance of this phenomenon wherein cytosine methylation is markedly dysregulated (normally hypomethylated loci shift to a methylated state). CIMP has been demonstrated in a number of different cancer types including hematological malignancies like AML. While methylation status has been studied predominantly at CpG islands, we used a novel assay (HELP; Khulan et al., Genome Res. 2006) to look for changes in cytosine methylation in large contiguous regions of the genome. We assessed global patterns of cytosine methylation by HELP analysis in a variety of tumor samples including leukemias and lymphomas. We found significant changes in the global methylation patterns of malignant cells, confirming prior observations of epigenetic dysregulation in these tumor types. We also discovered that the majority of the changes in cytosine methylation are occurring not at CpG islands but at other loci in the genome, including constitutively hypomethylated loci that we are finding to be candidate cis-regulatory sequences. We conclude that cytosine methylation changes in cancer occur much more extensively than analysis of CpG islands alone would indicate, and that the epigenetic dysregulation in cancer may be predominantly targeted to cis-regulatory sequences rather than to promoters.
BCL6 Programs Lymphoma Cells for Survival and Differentiation through Distinct Biochemical Mechanisms, Both of Which Can Be Therapeutically Targeted.
Authors: Parekh Samir , Polo Jose , Juszczynski Przemyslaw , Lev Paola , Ranuncolo Stella , Cattoretti Georgio , Shaknovich Rita , Shipp Margaret , Melnick Ari
Published: October 2019

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCL). Constitutive expression of BCL6 has been proposed to mediate lymphomagenesis through several mechanisms, including evasion of cell death, proliferation and differentiation blockade. We show here that BCL6 mediates these effects through distinct mechanisms. First, we show that blocking the association of the SMRT corepressor with BCL6 using our specific peptide inhibitor (BPI) abrogates only the survival effects of BCL6 but has no effect on differentiation. Accordingly, BPI upregulates survival genes such as ATR and p53, but not genes associated with differentiation such as Blimp1, XBP and Syndecan. In contrast, BCL6 shRNA upregulates both survival and differentiation genes and induces both cell death and differentiation. We and others have shown that BCL6 can also directly bind to the MTA3 corepressor, which is implicated in differentiation of Burkitt lymphoma cells. We found that BCL6 and MTA3 are co-expressed in DLBCL cells and primary human centroblasts (the precursor cell for most DLBCLs). The endogenous BCL6 and MTA3 proteins interacted in DLBCLs cells in co-immunoprecipitation experiments. In contrast to SMRT blockade with BPI, siRNA depletion of MTA3 induced expression of the Blimp1, XBP and Syndecan but not p53 and ATR. MTA3 depletion induced plasmacytic differentiation within 72 hours as shown in functional assays and by surface markers. We performed ChIP on chip using custom arrays densely tiling with oligonucleotides covering the entire genomic loci of 20 BCL6 target genes. Interestingly, BCL6 formed different types of repression complexes at differentiation genes (Complex with MTA3/NuRD) vs. survival genes (complex with SMRT and N-CoR). BCL6-mediated repression of genes involved in survival and differentiation thus depend on distinct biochemical mechanisms. The relevance of these findings for human disease was underscored by the fact that we found a statistically significant positive correlation between MTA3 and BCL6 gene expression in a database of 176 human DLBCLs (p<0.00001). Likewise, protein expression of BCL6 and MTA3 was also highly correlated (p<0.00001) (74 cases examined) and staining for Blimp1 revealed mutually exclusive expression from MTA3. Taken together these results illustrate the basic mechanisms through which BCL6 mediates DLBCL lymphomagenesis and provide the basis for powerful targeted therapy regimens that could be translated to the clinical setting.
DNA Methylation Inhibitors Upregulate MiRNA Expression in Patients with Chronic Lymphocytic Leukemia.
Authors: Yu Margaret K. , Mendell Joshua T. , Glenn Martha G. , Chen Zhong , Jones Kimberly A. , Phillips John D.
Published: October 2019

Chronic lymphocytic leukemia is a disease with a variable outcome. Since early treatment does not result in a survival benefit, clinicians often wait for evidence of progressive disease before offering systemic chemotherapy. We recently reported on the contribution of DNA methylation to disease progression. CLL patients with higher than age-expected global DNA methylation demonstrated a requirement for chemotherapy within 12 months as compared to patients with lower than age expected levels. We hypothesized that treatment of patients with early Rai Stage CLL can delay progression of disease by re-expression of tumor suppressor genes. We used two different DNA methylation inhibitors: a DNA methyltransferase enzyme inhibitor, 5-azacytidine, and a methyl substrate inhibitor, 2-chloro-2-deoxyadenosine. We demonstrate increased microRNA expression in six patients treated with 5-azacytidine and the one patient treated with 2-chloro-2-deoxyadenosine. In 6 out of 7 patients, DNA methylation globally decreased by 8% after treatment. However, consistent microRNA upregulation was seen in mir-17-3p, mir-21, mir-29a, mir-29b, mir-29c, mir-30e, mir-104, mir-126, mir-128a, mir-130a, mir-141, mir-142-3p, mir-148a, mir-151, mir-199a, mir-199a*, and mir-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Using the University of California Santa Cruz genome database, mir-17, mir-126, mir-128a, mir-148a, mir-151, mir-199a, and mir-301 are sequence conserved in at least 5 mammalian species and are associated with a CpG island upstream from the predicted transcriptional start site. All but mir-148a are embedded within a known gene. No changes were seen in bcl-2, p53, mir-15, or mir-16. Overexpression of mir-199-a and mir-199a* has been shown to induce cell cycle arrest. Mir-17-3p and mir-21 have anti-apoptotic properties. MiRNAs in patients with CLL are likely regulated by DNA promoter methylation.
High CLIP (Class II Associated Invariant Chain Peptide) Expression on Minimal Residual Disease Cells Is Associated with High Relapse Rate in AML Patients.
Authors: Chamuleau Martine E. , Feller Nicole , Kelder Angel , Van de Loosdrecht Arjan A. , Ossenkoppele Gert J.
Published: October 2019

MHC class II molecules are able to present tumor antigens to CD4+ T cells which play in their turn an important role in orchestrating the anti-tumor immune response. MHC class II molecules are only able to present antigen when the antigen binding site has been liberated from CLIP, which is a remnant of the intracellular chaperone molecule called Invariant Chain. Impaired MHC class II antigen presentation by persistent expression of CLIP on the antigen binding site of > 35% of the leukemic myeloid cells at diagnosis of AML patients is correlated to shorter overall survival (Chamuleau et al, Cancer Res.2004; 64:5546–5550). In this study, we investigated the CLIP expression on minimal residual disease (MRD) cells in the bone marrow of patients who achieved complete remission after chemotherapeutic treatment. Such expression may reveal the role of immune control in the outgrowth of MRD cells towards a relapse. The presence of MRD was measured by leukemia associated phenotype (LAP) expression (e.g. expression of the lymphoid cell marker CD7 or natural killer cell marker CD56 on myeloid blasts). Such aberrancies can be defined on AML blasts at diagnosis. At first, we established the percentage of MRD cells of 19 patients who remained in continuous complete remission (median follow up 44 months, range 7.4–109 months, sample taken at median 21 months after complete remission) and of 15 patients who relapsed soon after samples were taken (median time before relapse occurred: 5.9 months, range 1.4–20 months). The median frequency of MRD cells in relapsing patients was 0.1% (range 0.01 – 3.1%) compared to 0.02% (range 0.01–0.09%) in patients who remained in continuous remission. Differences were significant (p=0.017, Mann-Whitney U), in line with our previous finding that a high MRD cell frequency correlates with a high relapse rate (N. Feller et al, Leukemia2004, 18:1380–1390). Furthermore, we established the CLIP expression on the CD34+ LAP+ cells of both groups. Patients that relapsed showed a significant higher median CLIP expression (69%, range 6–96%, SD 27) than the patients that remained in continuous complete remission (median CLIP expression of 31.6% (range 5–93%, SD 26)) p=0.012 (Mann-Whitney U). In a logistic regression analysis both parameters were independent factors for predicting relapse (p=0.014 for CLIP expression and p=0.035 for MRD frequency). In conclusion, the impaired immunogenicity of low frequency MRD cells in patients that are morphologically in complete remission seems to play an important role in the outgrowth of MRD to relapsing leukemia. Enhancing the immunogenicity of MRD cells by restoration of the MHC class II antigen presentation pathway could offer new possibilities for immune therapy of AML patients.
The Role of C-KIT Mutations in the Leukemogenesis of Core-Binding Factor Acute Myeloid Leukemia: A Comparative Analysis on Paired Diagnosis and Relapse Samples.
Authors: Liang Der-Cherng , Shih Lee-Yung , Huang Chein-Fuang , Chang Ya-Tzu , Li Huei-Ying , Lai Chang-Liang
Published: October 2019

C-KIT is a member of the type III receptor tyrosine kinase family and plays a crucial role in normal hematopoiesis and acute myeloid leukemia (AML). C-KIT mutations have been described in core-binding factor (CBF) AML at initial diagnosis. The role of C-KIT mutations in the relapse of CBF AML is not clear. In the present study, we analyzed C-KIT mutations on paired diagnosis and relapse samples in CBF AML. Among 1014 adults and 162 children with AML, CBF AML was detected in 11.4% of adults and 25.3% of children. Mutational analysis of C-KIT was performed by direct sequencing for all cDNA PCR products amplified with 5 overlapping primer pairs, which cover the whole coding sequences of C-KIT gene from exon 1 through exon 21. In AML with t(8;21)/AML1-ETO, 33.0 % (29/88) of adults and 44.4 % (12/27) of children had C-KIT mutations. In AML with inv(16)/CBFβ-MYH11, 22.2 % (6/27) of adults and 38.5 %(5/13) of children had C-KIT mutations. Taken together, C-KIT mutations were present in 30.4 % (35/115) of adults and 42.5 % (17/40) of children with CBF AML. Forty-two patients with CBF AML relapsed. Twenty-two(18 adults and 4 children) of the 23 patients with CBF AML and C-KIT(+) at diagnosis had relapse samples available for comparative analysis. All the 22 patients relapsed with C-KIT mutations, 21 of them showed the identical C-KIT mutation patterns as those at diagnosis. Of the 20 relapsed patients with t(8;21)/AML1-ETO and C-KIT(+), 3 had mutations in exon 8: T417_D419delinsY, Y418_D419delinsA, and [Y418N;Y418_D419insFF], respectively; one had mutation in exon 9: I478V; another one had mutation in exon 11: [D572_P573insL; E561_D572dup]; 14 had mutations in exon 17: 5 D816V, 3 N822K, 3 D816Y, and one each with D816H, D820G, and D820Y; the remaining one patient relapsed twice, the patterns of C-KIT mutations changed but remained in exon 17: D816A at diagnosis, D816V at the first relapse, and N822K at the second relapse. Genotyping analysis with 15 loci of short tandem repeats at 13 different chromosomes showed identity for the diagnosis and the two relapse samples. Of the 2 adults with inv(16)/CBFβ-MYH11 and C-KIT(+) who relapsed, both had mutations in exon 17: N822K and D816Y, respectively. C-KIT mutations were absent in all of the 35 complete remission samples examined. In those with CBF AML and C-KIT(−) at diagnosis, 19 patients including 16 adults and 3 children relapsed; C-KIT mutations were not present in all the relapse samples except one who acquired D816H mutation. The present study showed that all patients with de novo CBF AML harboring C-KIT mutations at diagnosis retained the mutations at relapse, indicating that C-KIT mutations play a crucial role in the leukemogenesis in a substantial proportion of patients with CBF AML.
AML with 11q23/MLL Abnormalities: A Survey of 146 Cases from French AML Intergroup.
Authors: Banos Anne , Jaquet Antoine , Dombret Herve , Auvrignon Anne , Dastugue Nicole , Castaigne Sylvie , Leblanc Thierry , Fenaux Pierre , Rigal-huguet Francoise , Witz Francis , Blaise Didier , Reiffers Josy , Leverger Guy , Harousseau Jean-luc , Socie Gerard , Fiere Denis , Lioure Bruno , Bastard Christian , Thomas Xavier , Lamy Thierry , Pigneux Arnaud
Published: October 2019

Karyotype is one of the most important prognosis factor in acute myeloid leukemia. The MLL gene, located at 11q23, is fused to a variety of partner genes through chromosomal translocations and generates the critical leukemogenic fusion proteins. We performed a large retrospective study in 146 adults and children with 11q23/MLL + rearrangement enrolled in 6 clinical trials conducted in France between 1987 and 1998. Prognosis studies were performed in the 110 patients (47 adults and 63 children) who achieved a complete remission. Individual data were registred, including age, blood and marrow count, extramedullary disease and cytogenetics. The kaplan-Meier method was used in survival analysis and the cox proportinal-hazards model was used to analyse the effect of potential prognosis factors on survival. Among 47 adults, 43 % (20/47) carried t(9 ;11), 19% (9/47) carried t(6 ;11), 23% (11/47) carried t(11 ;19), 6% (3/47) carried t(10 ;11) and 8% (4/47) carried another abnormalities involved 11q23/MLL. Estimated 5-year disease-free survival (DFS), overall survival (OS) and relapse rate (RR) were respectively 17%, 21% and 77%. We analysed separately all translocations involved MLL and we found that the only translocation indicating a favorable prognosis was the translocation t(11 ;19). Compared with other translocations involving MLL, the estimated 5-year DFS, OS and RR of patients with a t(11 ;19) were respectively 45 vs 8% (p=.02), 54 vs 11% (p=.006) and 48 vs 84% (p=.04). Relative risk of relapse was 0,29 (95% confidence interval, 0.09 to 0,9 ;p=.03). Among 63 children, 63% (40/63) carried t(9 ;11), 3% (2/63) carried t(6 ;11), 8% (5/63) carried t(11 ;19), 14% (9/63) carried t(10 ;11) and 11% (7/63) carried another MLL rearrangement. Estimated 5-year DFS, OS and RR were respectively 59%, 72% and 36%. Compared with other translocations involving MLL, we found no difference in outcome between patients with t(9 ;11) and those with other rearrangement of MLL. In conclusion, the most common recurrent abnormality in this large study was translocation t(9 ;11) in adults and children. We found that adults patients whose leukemic cells contained the t(11 ;19) have a better outcome compared with those whose leukemic cells contain other 11q23 /MLL alterations. In children, we do not confirm the favorable impact of t(9 ;11) suggested in previous reports.