Deciphera Pharmaceuticals Research Articles

Vimseltinib versus placebo for tenosynovial giant cell tumour (MOTION): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial

Tenosynovial giant cell tumour (TGCT) is a locally aggressive neoplasm for which few systemic treatment options exist. This study evaluated the efficacy and safety of vimseltinib, an oral, switch-control, CSF1R inhibitor, in patients with symptomatic TGCT not amenable to surgery. MOTION is a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial done in 35 specialised hospitals in 13 countries. Eligible patients were adults (aged ≥18 years) with a histologically confirmed diagnosis of TGCT for which surgical resection could potentially worsen functional limitation or cause severe morbidity. Patients were randomly assigned (2:1) with interactive response technology to vimseltinib (30 mg orally twice weekly) or placebo, administrated in 28-day cycles for 24 weeks. Patients and site personnel were masked to treatment assignment until week 25, unless progressive disease was confirmed earlier. The primary endpoint was objective response rate by independent radiological review using Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST) at week 25 in the intention-to-treat population. Safety was assessed in all patients who received the study drug. The trial is registered with ClinicalTrials.gov, NCT05059262, and enrolment is complete. Between Jan 21, 2022, and Feb 21, 2023, 123 patients were randomly assigned (83 to vimseltinib and 40 to placebo). 73 (59%) patients were female and 50 (41%) were male. Nine (11%) of 83 patients assigned to vimseltinib and five (13%) of 40 patients assigned to placebo discontinued treatment before week 25; one patient in the placebo group did not receive any study drug. Objective response rate per RECIST was 40% (33 of 83 patients) in the vimseltinib group vs 0% (none of 40) in the placebo group (difference 40% [95% CI 29-51]; p<0·0001). Most treatment-emergent adverse events (TEAEs) were grade 1 or 2; the only grade 3 or 4 TEAE that occurred in more than 5% of patients receiving vimseltinib was increased blood creatine phosphokinase (eight [10%] of 83). One patient in the vimseltinib group had a treatment-related serious TEAE of subcutaneous abscess. No evidence of cholestatic hepatotoxicity or drug-induced liver injury was noted. Vimseltinib produced a significant objective response rate and clinically meaningful functional and symptomatic improvement in patients with TGCT, providing an effective treatment option for these patients. Deciphera Pharmaceuticals.

The Loss of ALDH9A1 Is a Significant Source of Endogenous DNA Damage Which May be Reversed By the Inhibition of Polyamine Transport System

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome and is caused by impaired DNA interstrand crosslink repair. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage likely play an important role in the pathogenesis of FA.We previously identified loss of ALDH9A1 as a significant source of endogenous DNA damage using a metabolism-focused CRISPR knockout (KO) screen. This finding was validated using Jurkat cells as well as human hematopoietic stem and progenitor cells. Here, we present updates of our project.To determine whether endogenous DNA damage was induced by the combined loss of FANCD2 and ALDH9A1, we investigated markers of DNA damage in bulk-edited cells. We found that the numbers of chromosomal breaks, 53BP1 foci, and gamma-H2AX foci were increased in FANCD2-/-ALDH9A1-/- cells, compared with single KO or wildtype (WT) controls, in the absence of an exogenous DNA damaging agent. These findings are consistent with spontaneously increased basal levels of DNA damage in FANCD2-/-ALDH9A1-/- cells.To study in vivo BMF and tumorigenesis phenotypes of ALDH9A1 deficiency in the setting of FA, we generated a mouse model. Fanca-/-Aldh9a1-/- mice showed the lowest frequency of long-term hematopoietic stem cells and lineage-negative Sca-1-positive cKit-positive cells, however the differences were not significant compared with control groups. While we did not observe aplastic anemia or leukemia, we found a higher incidence of solid tumors, most notably ovarian tumors and hepatocellular carcinoma in aged Fanca-/-Aldh9a1-/- mice. This suggests that the level of endogenous reactive aldehydes created by ALDH9A1 deficiency in mouse is not high enough to cause full-blown hematopoietic phenotypes, most likely due to redundant detoxification pathways. However, in tissues where ALDH9A1 is active and non-redundant, the low level of endogenous DNA damage accumulating over time causes solid tumors.To identify the specific reactive aldehydes responsible for DNA damage in ALDH9A1 deficiency, we performed a growth selection screen using FANCD2-/-ALDH9A1-/- cells. We found that the loss of ATP13A3 conferred survival advantage to FANCD2-/-ALDH9A1-/- cells. ATP13A3 transports endocytosed polyamines into the cytosol where polyamines can be metabolized by serum amine oxidases into 3-aminopropanal, a reactive aminoaldehyde. 3-aminopropanal also undergoes spontaneous decomposition to acrolein, a well-known reactive aldehyde carcinogen. Finding that the loss of ATP13A3 rescues that FANCD2-/-ALDH9A1-/- cells indicates that 3-aminopropanal and/or acrolein induces endogenous DNA damage requiring the Fanconi anemia pathway function for repair.Finally, to determine the contribution of ALDH9A1 variants to clinical manifestations of FA patients, we performed targeted sequencing of DNA from FA patients. We identified five missense variants, four of which had high CADD scores (>20). ALDH9A1 cDNA containing missense variants with high CADD scores expressed in ALDH9A1-/- Jurkat cells resulted in lower protein expression than the WT cDNA. Cell culture supernatant from cells expressing the variant cDNAs also had increased aldehyde levels as assessed by fluorometric assays, suggesting decreased enzymatic activity of the variant proteins. The patients with ALDH9A1 missense variants with high CADD scores had either early hematologic onset of FA (n=3; two patients before age 1 and one patient before age 4) or AML (n=1).In conclusion, we showed that the loss of ALDH9A1 generates endogenous DNA damage necessitating the FA pathway for its repair. Synthetic lethality caused by the combined loss of FANCD2 and ALDH9A1 was rescued by the loss of ATP13A3, which suggests that 3-aminopropanal is the culprit aminoaldehyde that accumulates in ALDH9A1-deficient cells and results in DNA damage. Functionally deleterious ALDH9A1 variants were observed in some FA patients with early onset of disease suggesting that ALDH9A1 could be a modifier of FA in humans. DisclosuresSridhar: Deciphera Pharmaceuticals: Current Employment; CRISPR Therapeutics: Ended employment in the past 24 months. White: Regeneron Pharmaceuticals: Current Employment. Smogorzewska: Rocket Pharmaceuticals: Research Funding.

Industry reveals a suite of new drug structures

Tucked away at the end of the first week of the American Chemical Society Spring 2021 meeting was the always-anticipated “First-Time Disclosures” session, in which industry reveals the structures of its latest clinical drug candidates. The event is a rare opportunity for chemists to peek inside the medicinal chemistry industry’s process for designing and homing in on new drugs. Chemists from Bristol Myers Squibb, Janssen Research and Development, and Deciphera Pharmaceuticals presented new cancer drugs. In addition, Ivan Efremov of Fulcrum Therapeutics presented a candidate that targets sickle cell disease (shown); Grace Chuang of Cytokinetics discussed a treatment for the rare genetic heart disease, obstructive hypertrophic cardiomyopathy; and Michael Siu from Genentech spoke about a drug for amyotrophic lateral sclerosis, commonly known as ALS. Session organizer Nicole Goodwin of GlaxoSmithKline pointed out that pyrazoles were popular this year, appearing in three of the six candidate molecules. “Pyrazoles for the win,”

Ripretinib: First Approval.

Ripretinib (QINLOCK™) is a novel type II tyrosine switch control inhibitor being developed by Deciphera Pharmaceuticals for the treatment of KIT proto-oncogene receptor tyrosine kinase (KIT)-driven and/or platelet derived growth factor receptor A (PDGFRA)-driven cancers, including gastrointestinal stromal tumour (GIST). Ripretinib inhibits KIT and PDGFRA kinase, including wild-type, primary and secondary mutations, as well as other kinases, such as PDGFRB, TIE2, VEGFR2 and BRAF. In May 2020, oral ripretinib received its first approval in the USA for the treatment of adult patients with advanced GIST who have received prior treatment with ≥ 3 kinase inhibitors, including imatinib. The US FDA, Health Canada and the Australian Therapeutic Goods Administration collaborated on the review of the ripretinib new drug application in this indication as part of Project Orbis; regulatory review in Australia and Canada is ongoing. Clinical development for GIST, solid tumours and systemic mastocytosis is underway in several countries worldwide. This article summarizes the milestones in the development of ripretinib leading to this first approval for the treatment of advanced GIST.

Combining the Allosteric ABL1 Inhibitor Asciminib (ABL001) with Ponatinib Suppresses Emergence of and Restores Efficacy Against Highly Resistant BCR-ABL1 Compound Mutants

Combining the Allosteric ABL1 Inhibitor Asciminib (ABL001) with Ponatinib Suppresses Emergence of and Restores Efficacy Against Highly Resistant BCR-ABL1 Compound Mutants

LBA87 - INVICTUS: A phase III, interventional, double-blind, placebo-controlled study to assess the safety and efficacy of ripretinib as ≥ 4th-line therapy in patients with advanced gastrointestinal stromal tumors (GIST) who have received treatment with prior anticancer therapies (NCT03353753)

LBA87 - INVICTUS: A phase III, interventional, double-blind, placebo-controlled study to assess the safety and efficacy of ripretinib as ≥ 4th-line therapy in patients with advanced gastrointestinal stromal tumors (GIST) who have received treatment with prior anticancer therapies (NCT03353753)

877P - Serum biomarkers of bone metabolism in metastatic castration-resistant prostate cancer (mCRPC) patients (pts) treated with radium-223 (Ra223): Results from a prospective multicentre study

BackgroundRa223 is a life-prolonging alpha-emitter bone targeted therapy for mCRPC patients with bone metastases. However, evidence on biomarkers that may help us in patient selection are lacking. Total ALP (tALP) appeared to be a potential marker of Ra223 effect in early studies (Sartor, Ann Oncol, 2017). Other bone-related markers, as bone-specific ALP (BALP), have demonstrated its prognostic value in mCRPC patients with bone metastases (Fizazi K, Eur Urol, 2015; Lara PN, J Natl Cancer Inst, 2014). MethodsPRORADIUM (NCT022925702) is a prospective multicentre cohort study in mCRPC patients treated with Ra223. The primary aim was to assess the impact of baseline serum biomarkers of bone formation (BALP and C-terminal of type 1 collagen propeptide [CICP]) on overall survival (OS). Secondary aims include the correlation of progression-free survival (PFS), time to PSA progression (TTPP) and skeletal-related events free-survival (SRE-FS) with serum bone markers. Results142 out of 168 pts enrolled in the study were included in this preliminary analysis. Median age was 74 yrs, 85.5% pts had ECOG 0-1, 52% pts completed 5-6 cycles of Ra223. Higher baseline levels of BALP and CICP were associated to number of metastases in bone-scan (p=0.007 and p=0.016, respectively) and baseline pain (p=0.014 and p=0.050, respectively). After a median follow-up of 18 months, 91 deaths were observed, with a median OS of 11.5 months (95%CI: 9.1-13.9). Patients with baseline BALP and CICP values above the median showed a trend to shorter TTPP (BALP: 2.8 vs 3.1 m, p=0.016; CICP: 2.8 vs 3.0 m, p=0.091) and PFS (BALP: 4.4 vs 5.1 m, p=0.070; CICP: 4.2 vs 5.5 m, p=0.281), respectively. The elevation of bone markers above the median was significantly associated with worse OS (BALP: 8.8 vs 17.9 m, p<0.001; CICP: 9.4 vs 16.8 m, p=0.001). There were not associations found with SRE-FS. ConclusionsOur results suggest that baseline serum markers of bone formation may serve as biomarkers for prognosis in mCRPC patients treated with Ra233. Clinical trial identificationNCT022925702. Legal entity responsible for the studyIBIMA and CNIO. FundingBayer, CRIS Cancer Foundation, Grant from Instituto de Salud Carlos III (PI16/01565). DisclosureN. Romero Laorden: Honoraria (self), Travel / Accommodation / Expenses: Bayer, Astellas Pharma, Janssen-Cilag, Sanofi-Aventis, PharmaMar, MSD, Roche. R. Lozano Mejorada: Honoraria (self): Roche, Janssen-Cilag, Bayer; Research grant / Funding (institution): Bayer, Janssen-Cilag; Travel / Accommodation / Expenses: Roche, Janssen-Cilag, Astellas Pharma. E. Almagro Casado: Honoraria (self): MSD; Travel / Accommodation / Expenses: BMS. E. Gonzalez Billalabeitia: Travel / Accommodation / Expenses: BMS, Pfizer, Janssen-Cilag, Astellas Pharma, Sanofi. A. Montesa: Advisory / Consultancy: Janssen-Cilag, Pfizer, Sanofi, Astellas Pharma; Travel / Accommodation / Expenses: Pfizer. G.A. De Velasco Oria de Rueda: Honoraria (self), Advisory / Consultancy: Pfizer, Novartis, Ipsen, Astellas Pharma, BMS, Bayer, MSD, Roche. R. Morales Barrera: Honoraria (self): MSD, Sanofi, AstraZeneca, Janssen, Roche/Genentech; Advisory / Consultancy: MSD, Sanofi, AstraZeneca, Janssen, Roche/Genentech; Speaker Bureau / Expert testimony: MSD, Sanofi, AstraZeneca, Asofarma, Janssen; Research grant / Funding (institution): AB Science, Aragon Pharmaceuticals, INC, Astellas Pharma, AstraZeneca, Aveo Pharmaceuticals, Bayer, Blueprint Medicines Corporation, BN Immunotherapeutics, Boehringer Ingelheim España, BMS, Clovis Oncology, Cougar Technology, Deciphera Pharmaceuticals LL; Travel / Accommodation / Expenses: MSD, Roche, Lilly, Clovis Oncology, Bayer, Janssen-Cilag, Astellas Pharma, AstraZeneca. D. Lorente: Honoraria (self): Janssen-Cilag, Bayer, Astellas Pharma, Sanofi; Advisory / Consultancy: Janssen-Cilag, Bayer, Sanofi; Travel / Accommodation / Expenses: Sanofi, Astellas, Janssen-Cilag, Celgene. E. Castro: Honoraria (self): Astellas Pharma, Janssen-Cilag, AstraZeneca, Bayer, Pfizer; Advisory / Consultancy: Bayer, Janssen-Cilag; Research grant / Funding (institution): Janssen-Cilag, AstraZeneca, Bayer, Genentech, Roche, Pfizer, Astellas Medivation, Tokai Pharmaceuticals; Travel / Accommodation / Expenses: Bayer, Janssen-Cilag, Roche, Astellas Pharma. D. Olmos Hidalgo: Honoraria (self): Janssen-Cilag, Bayer, Sanofi; Advisory / Consultancy: Bayer, Janssen-Cilag, AstraZeneca, Clovis Oncology; Research grant / Funding (institution): Bayer, Janssen, AstraZeneca, Roche/Genentech, Medivation/Pfizer, Astellas, Tokai, MSD, GSK; Travel / Accommodation / Expenses: Bayer, Janssen-Cilag, Ipsen. All other authors have declared no conflicts of interest.

Frequency and Prognostic Significance of Cytogenetic Abnormalities in 1269 Patients with Therapy-Related Myelodysplastic Syndrome - a Study of the International Working Group (IWG-PM) for Myelodysplastic Syndromes (MDS)

Frequency and Prognostic Significance of Cytogenetic Abnormalities in 1269 Patients with Therapy-Related Myelodysplastic Syndrome - a Study of the International Working Group (IWG-PM) for Myelodysplastic Syndromes (MDS)

Evaluation of Cell Surface Markers and Targets in Leukemic Stem Cells (LSC) Reveals Distinct Expression Profiles, Unique Drug Effects, and Specific Checkpoint Regulation in AML LSC and CML LSC

In an attempt to identify novel cell surface markers and targets in leukemic stem cells (LSC) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples of patients with AML (n=257), CML (n=134), and controls (n=256: other BM neoplasms, n=116; normal/reactive BM, n=29; idiopathic cytopenia, n=31; lymphoma patients without BM involvement, n=80) for expression of cell surface markers and targets on CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells (PC) by multi-color flow cytometry. In addition, we examined mRNA expression profiles in highly purified CD34+/CD38− stem cells and CD34+/CD38+ PC by gene array- and qPCR analyses. Aberrant LSC expression profiles were identified in all patients examined. In patients with CML, CD34+/CD38− LSC expressed an almost invariable aberration profile defined as CD25+/CD26+/CD56+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably displayed aberrant surface antigens, including CD25 (55%), CD96 (35%), CD371 (CLL-1) (75%), and IL-1RAP (60%). With the exception of a subset of FLT3 ITD+ patients (45% of all FLT3-mutated cases), AML LSC did not exhibit CD26. All other markers and targets identified on AML and/or CML LSC, including CD9, CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, neural proliferation and differentiation control-1 antigen (NPDC1), and roundabout-4 (ROBO4), were also detectable on normal hematopoietic stem cells (HSC). However, several surface-targets, including CD33 and CD52, were expressed at higher levels on CD34+/CD38− LSC compared to normal HSC, and antibody-mediated targeting resulted in their selective depletion and in a significantly reduced engraftment of LSC in NSG mice. Since certain surface antigens, like CD47 (IAP), CD243 (MDR1), and CD274 (PD-L1) may contribute to intrinsic drug resistance of LSC, we also examined the expression of these antigens in AML and CML LSC. Regardless of the type of leukemia, LSC were found to express CD47 in all patients examined. MDR-1 was found to be expressed on LSC in 11/39 patients with AML (28%) and 2/22 patients with CML (9%). PD-L1 was not detectable on LSC in untreated BM samples. Exposure of LSC to interferon-gamma (IFN-G, 100 U/ml, 48 hours) resulted in expression of PD-L1 on LSC in all patients with Ph+ CML (control: 100%; IFN-G: 183±40%, p<0.05). Unexpectedly, however, IFN-G was found to induce PD-L1 expression in only 1 out of 11 AML patients tested. IFN-G showed no effects on expression of CD47 or MDR-1 in LSC. We next examined the potential mechanisms of IFN-G-mediated expression of PD-L1 on LSC and screened for drugs capable of counteracting the expression of this resistance-inducing checkpoint molecule. Of all targeted drugs examined (n=10) only the BRD4/MYC inhibitor JQ1 was identified as a regulator of IFN-G-induced PD-L1 expression on CML LSC. In particular, JQ1 (2.5 µM, 48 hours) was found to suppress IFN-G-induced (100 U/ml, 48 hours) upregulation of PD-L1 in primary CML LSC in all donors tested (IFN-G: 183±40% versus IFN-G+JQ1: 136±27% of control) suggesting that checkpoint expression in LSC is regulated by epigenetic mechanisms and MYC activity. Together, we have established cell surface marker- and target expression profiles in CD34+/CD38− AML LSC and CML LSC which should facilitate their enrichment and may support the development of LSC-eradicating treatment concepts. In addition, the unique expression profiles of LSC should provide a powerful basis for diagnostic LSC phenotyping in patients with CML and AML. DisclosuresHoermann:Novartis: Honoraria; Gilead: Research Funding; Ariad: Honoraria; Amgen: Honoraria. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Valent:Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria.

Incidence and Prognostic Impact of Cytogenetics in Combination with Molecular Aberrations in Patients with Systemic Mastocytosis

Systemic mastocytosis (SM) usually arises as a consequence of an acquired mutation in the receptor tyrosine kinase KIT, usually KIT D816V (>80-90% of patients). The presence of additional molecular aberrations, e.g. in SRSF2, ASXL, or RUNX1 (S/A/Rpos), has a strong adverse impact on disease phenotype and prognosis in patients with advanced SM (advSM) (Jawhar et al., Leukemia 30, 2016). However, little is known about the frequency and prognostic impact of cytogenetic aberrations and potential interactions between cytogenetic and molecular aberrations in the context of prognostication. We therefore analyzed clinical, morphological, cytogenetic and molecular characteristics of 109 patients (KIT D816V, n=102, 94%; KIT D816H, n=2, 2%; no KIT mutation, n=5, 5%) with indolent SM (ISM, n=27) and advSM (n=82) with or without an associated hematologic neoplasm (AHN) [SM-AHN, n=60; aggressive SM, n=3; mast cell leukemia (MCL), n=9; MCL-AHN, n=10)]. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes as previously described (Schwaab et al., Blood 122, 2013). All ISM patients had a normal karyotype. In advSM, an aberrant karyotype was identified in 16/82 (18%) patients. By analogy with other myeloid neoplasms, e.g. MDS or AML, patients were classified according to their karyotype into two groups. The good-risk group (n=73) included patients with a normal karyotype (n=66) plus those with a favorable karyotype (n=7; del(5q), n=4; trisomy 8, n=1; del(1q), n=1; del(12p), n=1), while the poor-risk group (n=9) included patients with a complex karyotype (defined as ≥3 abnormalities; n=8) or monosomy 7 (n=1). Seven of 9 (78%) patients with a poor-risk karyotype and 2/7 (29%) patients with a favorable karyotype progressed to secondary AML (n=7) or MCL (n=2) within a median observation time of 13 months (range, 0-26). Overall, the median overall survival (OS) of patients with a poor-risk karyotype was significantly shorter than in patients with a good-risk karyotype (4 vs. 41 months; hazard ratio, HR, 9.6, 95% CI 3.9-23.2; P<0.0001). Of the 67 cases that were tested for gene mutations 5/9 (56%) with a poor-risk karyotype and 31/58 (53%) patients with a good-risk karyotype were S/A/Rpos. Significant differences regarding OS were observed when comparing the following 3 groups (Figure): good-risk karyotype + S/A/Rneg (n=27) vs. good-risk karyotype + S/A/Rpos (n=31) vs. poor-risk karyotype (n=9). In 6 multi-mutated advSM patients with simultaneous occurrence of cytogenetic [del(5q), n=3; trisomy 8, n=3], we explored the clonal architecture in single-cell-derivedgranulocyte-macrophage colonies (CFU-GM; median number per patient, n=12; range 10-15) using fluorescence in situ hybridization and mutation-specific PCR followed by Sanger sequencing. None of the colonies were positive for both KIT D816V and the cytogenetic aberration, indicating that these abnormalities marked different clones. However, some cytogenetically positive colonies were tested positive for additional mutations such as JAK2 V617F or TP53. In colonies without cytogenetic aberrations, additional mutations, e.g. SRSF2, ASXL1, RUNX1 or TET2, were identified with or without KIT D816V, confirming our previous results that the KIT D816V mutation is not the initial (earliest) event in SM evolution (Jawhar et al., Leukemia 29, 2015). We conclude, that a) molecular and cytogenetic analyses should be routinely performed in patients with advSM in addition to clinical and morphological baseline staging because they may more accurately indicate risk of disease progression/transformation and poor prognosis and b) patients with SM-AHN and an aberrant karyotype frequently harbor two different clones, with one responsible for SM evolution and the other driving AHN evolution independent of KIT D816V. [Display omitted] DisclosuresValent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

The Genomic and Transcriptomic Landscape of Systemic Mastocytosis

Background and AimSystemic mastocytosis (SM) is characterized by a heterogeneous spectrum of disease variants with different clinical features and prognosis. In indolent SM (ISM), the clinical course is usually stable and patients (pts) have a normal life expectancy. In contrast, aggressive SM (ASM) and mast cell leukemia (MCL) are characterized by severe organ damage and are fatal forms with short survival times. All forms of SM share the presence of activating mutations in the KIT tyrosine kinase (most frequently, the D816V mutation), which suggests that cooperating events might be responsible for the profoundly different clinical presentation and outcome of advanced SM (ASM and MCL) as compared to ISM. However, no robust studies using genome-wide approaches to scan for additional molecular aberrations in ASM and MCL have been published to date. This prompted us to use exome and RNA sequencing and single nucleotide polymorphism (SNP)-arrays to search for novel (and potentially druggable) molecular lesions.MethodsSeven pts with MCL, 6 pts with ASM and 2 pts with ISM were studied using whole exome sequencing (WES) and single nucleotide polymorphism (SNP)-arrays. WES (80x) was performed on a Hiseq 2500 (Illumina). SNP-arrays were done using Cytoscan HD Arrays (Affymetrix). Matched normal/mast cell DNA was analyzed in all but 2 archival MCL cases for whom no germline DNA source was available. RNA sequencing was also performed in 3/7 MCL and 1/6 ASM cases for whom high quality RNA was available. Data analysis and integration was performed according to in-house optimized pipelines.ResultsSNP-arrays found arm-level copy number (CN) or copy-neutral loss-of-heterozygosity (CN-LOH) events in 5/7 MCL pts (loss of 7p, 8p, 9q, 12p, 13q and CN-LOH at 4q, 18q and 21q). In contrast, ASM and ISM showed only focal CN and CN-LOH events. Overall, a median of 28 (range, 20-78) and 77 (range, 55-132) submicroscopic CN and CN-LOH events, respectively, were detected. The number of events did not significant differ between ISM, ASM and MCL. A total of 54, 102 and 1835 genes were significantly found to be recurrently involved in loss, gain and CN-LOH events, respectively. The average number of nonsynonymous mutations identified by WES was 15 in ISM and 35 in ASM and MCL. Signature analysis showed that the majority of sequence changes were C>T transitions and C>A transversions. Among genes recurrently mutated in ≥2 patients or concurrently identified by WES and SNP-arrays or known to be associated with cancer, some had already been reported to be mutated in SM: ASXL1, TET2, CBL, RUNX1, NRAS, IDH1, SRSF2, SF3B1. Alterations were also found in genes not previously implicated in SM, including RUNX3, MLL2, MLL3, CDC27, TP53BP1, CCND3, NCOR2, BCORL1, ATM, WRN, ARID1B, ARID3B, KDM1B, ARID4A, SETD1A, SETD1B, PRDM1. Pathway analysis showed enrichment of genetic lesions affecting the following cellular processes: PI3K/Akt and MAPK pathways, calcium signaling, chromatin modification, DNA methylation and DNA damage repair. RNA-seq results, filtered against those of a set of normal bone marrow samples, revealed a high number of RNA chimeras involving two adjacent genes in the same transcription orientation and invariably joining the pre-last exon of the upstream gene to the second exon of the downstream gene - a fusion pattern suggestive of intergenic splicing following transcription read-through events. Deletions at the intergenic region was excluded by SNP-arrays for all the predicted transcription-induced chimeras. In addition, in all the 4 pts analyzed, retention of some or all introns in mature transcripts was observed for some genes, including known tumor suppressors (TS) and other key genes like TP53, BAX, ERCC2, WRN, FANCD2, ATR; U2AF2, U2AF1, SRSF6.Conclusions•SNP-array and WES analysis of SM revealed a heterogeneous landscape of molecular alterations, although some key cellular pathways were found to be recurrently altered.•More interestingly, RNA-seq uncovered perturbation of transcript elongation and splicing in all the advanced SM pts analyzed. Intron retention, in particular, seems to be a novel recurrent mechanism of TS gene inactivation at the posttranslational level in advanced SM. The mechanisms leading to intron retention and their cellular consequences deserve further investigation.Supported by AIRC (project code 16996), progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresSoverini:Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Cavo:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Martinelli:MSD: Consultancy; Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau.

Combined Targeting of STAT3 and STAT5: A Novel Approach to Overcome Drug Resistance in Ph+ Cml

Chronic myeloid leukemia (CML) is a stem cell disease characterized by BCR-ABL1. Most patients in chronic phase (CP) CML achieve long-lasting molecular responses when treated with BCR-ABL1 tyrosine kinase inhibitors (TKI). However, resistance against TKI occurs in a subset of patients. Several molecular mechanisms, including BCR-ABL1 mutations, contribute to TKI resistance. For imatinib-resistant patients, second- and third generation TKI, including nilotinib, dasatinib, bosutinib, and ponatinib, are available. Using these drugs, it is now possible to cover most of the known BCR-ABL1 mutations, including the multi-resistant mutation T315I. Ponatinib, a third generation TKI, induces growth-inhibitory effects in drug-resistant patients even if T315I is expressed. However, not all mutant forms of BCR-ABL1 are responsive to ponatinib. Moreover, it has been described that multiple secondary mutations in BCR-ABL1, especially T315I-involving compound mutations, confer resistance against ponatinib. Furthermore, resistance against TKI may develop independent of BCR-ABL1 mutations. Therefore, drug combinations covering a broad range of targets, are currently under investigation with the aim to overcome drug resistance in advanced CML. Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid that has been described to induce ROS generation and to suppress a number of survival-related molecules, including AKT, mTOR, and STAT3. The aim of the current project was to evaluate the anti-leukemic effects of CDDO-Me in TKI-resistant CML cells. As assessed by 3H-thymidine uptake experiments, CDDO-Me was found to inhibit growth of various CML cell lines, including K562, an imatinib resistant sub-clone of K562, KU812, and imatinib-resistant KCL22 cells (IC50: 0.1-0.5 µM). These effects were accompanied by induction of apoptosis as assessed by staining for AnnexinV and propidium iodide. Furthermore, CDDO-Me was found to block the growth of Ba/F3 cells harboring the BCR-ABL1 mutations T315I, E255K, G250E, H396P, or F359V as well as Ba/F3 cells expressing TKI-resistant compound mutations, such as T315I/E255V, T315I/F311L, T315I/F359V, or T315I/G250E (IC50: 0.1-0.25 µM). The anti-proliferative effects of CDDO-Me were also confirmed in primary CML cells isolated from 13 patients with chronic phase (CP) CML (4 TKI-resistant patients, 3 with BCR-ABL1 mutations), one in blast phase (BP), and one suffering from ponatinib-resistant Ph+ ALL harboring BCR-ABL1T315I/E255K. IC50 values were comparable between samples isolated from freshly diagnosed patients (IC50: 0.1-0.5 µM) and samples isolated from heavily pre-treated patients, (IC50: 0.1-0.5 µM) suggesting that BCR-ABL1 mutations do not influence responses to this drug. In consecutive experiments, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with second- or third-generation BCR-ABL1 TKI. The combination ´CDDO-Me+ponatinib´ was found to be effective in Ba/F3 cells expressing various BCR-ABL1 mutations, including T315I-involving compound mutations. We also found that the combination ´CDDO-Me+TKI´ leads to simultaneous dephosphorylation of STAT3 and STAT5. To clarify whether this drug action contributes to the synergistic drug-interactions observed, we performed experiments with shRNA directed against STAT3 or STAT5 and the specific STAT5-inhibitor AC-3-019. Knockdown of STAT3 was found to produce synergistic effects with TKI and with AC-3-019 in K562 and KCL22 cells, whereas STAT5-knockdown sensitized CML cells against CDDO-Me, pointing to a new effective concept of dual STAT3+STAT5 inhibition. However, CDDO-Me was also found to increase expression of heme-oxygenase-1 (HO-1), a heat-shock-protein known to trigger drug resistance and cell survival in CML cells. We therefore combined CDDO-Me with the HO-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects in human CML cells and BCR-ABL1+Ba/F3 cells. Moreover, SMA-ZnPP was found to sensitize KU812 cells and Ba/F3 cells expressing BCR-ABL1T315I/F311L against the combination ´CDDO-Me+TKI´. Together, combined targeting of STAT3, STAT5, and HO-1 overcomes multiple forms of TKI resistance in highly resistant CML clones expressing BCR-ABL1T315I or T315I-containing compound mutations. Whether such drug combinations are effective in vivo in TKI-resistant patients remains to be elucidated. DisclosuresHoermann:Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria; Novartis: Honoraria. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Deininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lion:Amgen: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Valent:Ariad: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Amgen: Honoraria.

Hydroxyurea Induces Growth Inhibition in BCR-ABL1 T315I+ Clones and Synergizes with Ponatinib in Killing TKI-Resistant CML Cells

In chronic myeloid leukemia (CML), the occurrence of BCR-ABL1 T315I is associated with resistance against first- and second-generation BCR-ABL1 tyrosine kinase inhibitors (TKI). Ponatinib is a third generation TKI that exerts strong anti-neoplastic effects in advanced CML and is capable of suppressing the kinase activity of BCR-ABL1 T315I. However, therapy with ponatinib is associated with potentially severe side effects. In addition, resistance against ponatinib may develop in sub-clones carrying multiple (compound) mutations in BCR-ABL1. In addition, BCR-ABL1-independent oncogenic pathways contribute to drug resistance. For these patients, alternative therapies such as stem cell transplantation (SCT) or various drug combinations are often considered.Hydroxyurea (HU) is used for initial or palliative cytoreduction in CML. However, the effects of HU on TKI-resistant mutant sub-clones have not been examined so far. The aims of this study were to explore the effects of HU on CML clones carrying BCR-ABL1 T315I as individual mutation or in compound-context, and to investigate anti-leukemic effects of the drug combination ponatinib+HU. In in vitro studies, primary patient-derived cells, human CML cell lines (K562, KU812, KCL-22), and Ba/F3 cells expressing wild type (wt) BCR-ABL1, BCR-ABL1 T315I, or BCR-ABL1 compound mutants involving T315 were examined. Cell proliferation was quantified by measuring 3H-thymidine uptake. Drug effects on competitive clonal growth were analyzed by mixing two Ba/F3 clones, one expressing BCR-ABL1 T315I with GFP and one BCR-ABL1 T315I/E255V labeled by tdTomato, at a 1:1 ratio. Then, cells were exposed to HU, pontinib, or HU+ponatinib for 72 hours, and the percentage of viable cells in each clone was analyzed by flow cytometry. The in vivo response of primary CML cells carrying BCR-ABL1 T315I to HU was examined in 4 TKI-resistant CML patients who were treated with HU (1-3 g/day) for up to 18 months. In these patients, we measured white blood counts (WBC), differential counts, and BCR-ABL1 transcript levels in peripheral blood (PB) by qPCR. The percentage of BCR-ABL1 T315I compared to total BCR-ABL1 was determined by ligation-dependent PCR. In all 4 patients treated with HU, WBC and total BCR-ABL1 mRNA levels remained stable for 3-12 months. Surprisingly, in 3 of 4 patients, the leukemic sub-clone expressing BCR-ABL1 T315Iwas no longer detectable after HU-treatment. After 3 months, 2/4 patients received allogenic SCT. In the other 2 patients, the disease remained stable for 6 and 12 months, respectively. In our in vitro studies, HU was found to inhibit the growth of all BCR-ABL1+ cell lines, including K562 (IC50: 1120±89 µM), KU812 (IC50: 216±32 µM), and KCL-22 (IC50: 196±23 µM) as well as Ba/F3 cells harboring BCR-ABL1 T315I as single mutation (IC50: 74±25 µM) or as compound together with E255V (IC50: 86±2 µM), F311L (IC50: 76±20 µM), F359V (IC50: 69±10 µM), or G250E (IC50: 89±4 µM). Interestingly, Ba/F3 cells exhibiting BCR-ABL1 T315I alone or in compound configuration were more sensitive to HU compared to Ba/F3 cells expressing wt BCR-ABL1 (IC50: 236±49 µM). As expected, HU was also found to inhibit growth of primary CML cells. In subsequent experiments, HU and ponatinib were found to synergize with each other in inhibiting growth of K562, KU812, and KCL-22 cells as well as Ba/F3 cells carrying BCR-ABL1 T315I (Figure) or BCR-ABL1-T315I/F359V. In cell mix experiments, ponatinib exerted strong growth-inhibitory effects on Ba/F3-T315I cells but not on Ba/F3-T315I/E255V cells, whereas HU was found to produce stronger effects on Ba/F3-T315I/E255V cells, and only the combination of both drugs resulted in complete suppression of both cell lines. In conclusion, HU exerts strong sub-clone-specific anti-neoplastic effects in TKI-resistant CML cells, both in patients with BCR-ABL1 T315I+CML and in various cell line models, including sub-clones harboring BCR-ABL1 T315I as single mutation or in compound configuration. In addition, we show that HU and ponatinib produce strong synergistic anti-neoplastic effects on TKI resistant CML cells, including sub-clones carrying T315I. These observations may have clinical implications and may pave the way for more effective sub-clone-eradicating but also palliative or bridging-to-SCT concepts in advanced CML. Clinical studies are now warranted to define the exact value of the drug combination ponatinib+HU in TKI-resistant CML. [Display omitted] DisclosuresSperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Lion:Ariad: Honoraria; Amgen: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Hoermann:Ariad: Honoraria; Novartis: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

The Multi-Kinase Inhibitor DCC-2618 Inhibits Proliferation and Survival of Neoplastic Mast Cells and Other Cell Types Involved in Systemic Mastocytosis

▪Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and pathologic accumulation of neoplastic mast cells (MC) in various internal organs. The indolent variant of SM (ISM) is associated with an almost normal life expectancy. By contrast, the prognosis in advanced SM, including SM with an associated hematologic neoplasm (AHN), aggressive SM (ASM), and MC leukemia (MCL) is poor with short survival times. Most patients with SM express the D816V-mutated variant of KIT, which confers resistance against several tyrosine kinase inhibitors (TKI), including imatinib. Midostaurin is a TKI that is effective against KIT D816V. However, despite encouraging clinical efficacy, this drug cannot produce continuous complete remission in all patients. One problem in advanced SM is that the AHN component of the disease, especially when progressing into acute myeloid leukemia (AML) is often drug-resistant. The aims of this study were to evaluate the effects of the multi-kinase inhibitor DCC-2618 on proliferation and survival of primary neoplastic mast cells, various mast cell lines and other malignant and non-malignant cell types that may play a role in advanced SM. As assessed by 3H-thymidine-uptake, DCC-2618 was found to inhibit the proliferation of all human MC lines tested, with lower IC50 values measured in HMC-1.1 cells lacking KIT D816V (11.2±4.3 nM) and ROSAKIT WT cells (61±11 nM) than in KIT D816V+ HMC-1.2 cells (147±68 nM) and ROSAKIT D816V cells (133±43 nM). DCC-2618 also produced growth inhibition in the multi-resistant MCL lines MCPV-1.1 (164±72 nM), MCPV-1.2 (256±167 nM), MCPV-1.3 (124±46 nM), and MCPV-1.4 (235±114 nM). In addition, DCC-2618 was found to inhibit the proliferation of primary neoplastic bone marrow MC obtained from patients with SM including MCL (Figure). We also found that DCC-2618 induces apoptosis in HMC-1 cells and ROSA cells, and to a lesser degree in MCPV-1 cells as determined by light microscopy and AnnexinV/PI staining. Moreover, DCC-2618 was found to block phosphorylation of KIT in all MC lines tested. In a next step, we explored the effects of DCC-2618 on growth of other leukemia cell lines as well on vascular endothelial cells. In these experiments, we were able to show that DCC-2618 inhibits the proliferation of the FIP1L1-PDGFRA+ eosinophilic leukemia cell line EOL-1 (IC50 2±0.6 nM) and the FLT3 ITD-mutated AML cell lines MV4-11 (IC50 130±18 nM) and MOLM-13 (IC50 110±26 nM). DCC-2618 also induced apoptosis in EOL-1, MV-411, and MOLM-13 cells. Moreover, DCC-2618 was found to inhibit the growth of cultured human vascular endothelial cells, suggesting that the drug may also counteract SM-related neo-angiogenesis in SM. DCC-2618 did not inhibit the proliferation of the immature AML cell line KG1 and the monoblastic cell line U937, but was found to block proliferation in primary leukemic monocytes in patients with monoblastic AML or chronic myelomonocytic leukemia (CMML) which may have clinical implications as CMML and AML are the most prevalent types of AHN in advanced SM. Finally, we were able to show that the major DCC-2618-metabolite, DP-5439, is equally effective in producing growth inhibition in all cell lines tested as well as in primary neoplastic MC compared to DCC-2618 (Figure). In summary, our data show that DCC-2618 is a new potent multi-targeted TKI that counteracts growth of neoplastic MC as well as growth and survival of leukemic monocytes, AML blasts, eosinophils, and endothelial cells in vitro. Whether DCC-2618 is also able to inhibit the growth of neoplastic MC and other leukemic (AHN) cells in vivo in patients with advanced SM remains to be determined in clinical trials. Indeed, a first Phase I clinical trial examining the effects of DCC-2618 in SM has recently been initiated. [Display omitted] DisclosuresValent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding.

Intensive Post-Remission Consolidation with G-CSF-Support: Tolerability, Safety, Reduced Hospitalization, and Efficacy of a New Treatment Protocol for AML Patients ≥60 Years

Treatment of AML in elderly patients (≥60 years) deserves special considerations and has to balance toxicity against efficacy as well as life expectancy against the risk of AML-related death and relapse after intensive treatment. We have previously shown that induction chemotherapy followed by repetitive cycles of intermediate dose ARA-C (IDAC) is a well-tolerated and highly effective approach for elderly patients with AML (Sperr et al, Clin Cancer Res 2004;10:3965-3971). The aim of the current multi-center study (EudraCT number: 2007-005806-29) was to evaluate the efficacy and feasibility of intensified consolidation therapy employing FLAG (cycle 1; fludarabine, 30 mg/m² iv, days 1-5, ARA-C, 2000 mg/m² iv, days 1-5) and IDAC (cycles 2-4; ARA-C, 2x1,000 mg/m2 iv, days 1,3,5) in elderly AML patients and to analyze the effect of pegfilgrastim (6 mg s.c. on day 6) on the duration of neutropenia, overall toxicity, and hospitalization-time during consolidation in these patients. In addition, overall survival (OS) and relapse-free survival were captured. To evaluate the efficacy of pegfilgrastim we compared consolidation cycles where pegfilgrastim was given routinely on day 6 (IDAC-P; consolidation 2 and 4) with cycles where pegfilgrastim was only used in case of infections and/or prolonged neutropenia (consolidation 3). Between Jul/2008 and Nov/2012 (observation period until Sept/2014) 64 patients with de novo AML were included (median age: 69.9 years; range 60.1- 85.2 years; 53 patients < 75 years, 11 patients ≥75 years; f:m ratio 1:1.56). Following induction therapy, 43 patients (68%) entered complete remission (CR), 33 after the 1st (DAV), 9 after a 2nd (MiDAC light: ARA-C 2x1g/m², days 1,3,5; mitoxantron 12 mg/m², days 3,5), and 1 patient after a 3rd (FLAG) induction cycle. In 16 patients no remission (NR) was achieved. Of these, 7 received only one induction cycle, 3 two, and 6 three cycles. Two patients died and 2 patients were withdrawn from the study during the 1st induction. Thirty-nine CR-patients (91%) were eligible for consolidation therapy. All 4 planned consolidation cycles could be administered in 23/39 patients (59.0%), 5/39 (12.8%) received 3 cycles, 3/39 (7.7%) 2 cycles, and 8/39 (20.5%) 1 cycle of consolidation therapy. Relapse (n=8; 20.5%) was the major cause of withdrawal, followed by persistent cytopenia (n=4; 10.2%), severe infections (n=2; 5.1%), and stem cell transplantation (n=1). Four patients died in the consolidation phase (treatment related, n=2; not treatment related, n=2). In 17 patients the efficacy of pegfilgrastim could be evaluated. The median duration of severe neutropenia (ANC<500/µL) was 7 days (range: 3-14 days) in the 2nd (IDAC-P), 12 days (range: 3-21 days) in the 3rd (IDAC), and 7.5 days (range: 5-19 days) in the 4th (IDAC-P) consolidation cycle. Differences in the duration of severe neutropenia between consolidation 2 and 3 and between consolidation 3 and 4, were significant (p<0.05). The duration of hospitalization differed also significantly among these patient-groups, with 21 days (range 13-42) in consolidation 2 (IDAC-P), 28 days (range 7-41) in consolidation 3 (IDAC), and 22 days (range: 9-40) in consolidation 4 (IDAC-P) (p<0.05). The median OS, continuous complete remission and disease-free survival were 1.1, 1.3, and 1.2 years respectively. The probability to be alive after 5 years was 32% (Figure). The median OS differed significantly between patients aged <75 years (1.5 years) and ≥75 years (0.5 years) (p<0.05) (Figure). Together, our data show that intensified consolidation can be safely administered in AML patients aged ≥60 years. Patients aged <75 years seem to benefit from this strategy. Moreover, the administration of pegfilgrastim on day 6 in IDAC consolidation cycles shortened the duration of neutropenia and the duration of hospitalization significantly. [Display omitted] DisclosuresSperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Knoebl:Baxalta, now part of Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Jaeger:Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Geissler:Novartis: Honoraria. Valent:Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Amgen: Honoraria.

Identification of CD44 As a RAS-MEK-Regulated Invasion Receptor That Is Overexpressed in Neoplastic Mast Cells and Triggers Disease Expansion in Advanced Systemic Mastocytosis

CD44, also known as Hermes antigen, is a multifunctional invasion receptor that mediates homing and expansion of normal and neoplastic stem- and progenitor cells in various organs including the bone marrow (BM). Mast cells (MC) and their progenitors also express CD44. However, little is known about the impact and regulation of CD44 in neoplastic cells in systemic mastocytosis (SM).We examined the expression, regulation, and functional role of CD44 in neoplastic cells in SM. As assessed by multi-color flow cytometry, CD34+/CD38- stem cells (SC), CD34+/CD38+ progenitor cells (PC), and KIT+/CD34- MC invariably expressed CD44 in all SM variants, including patients with indolent SM (ISM, 11/11), SM with associated hematologic neoplasm (SM-AHN, 7/7), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 8/8). Expression of CD44 on SC, PC, and MC increased significantly with the aggressiveness of SM. Moreover, soluble CD44 levels measured in the sera of patients with SM by ELISA were found to correlate with the WHO type of SM. In particular, significantly higher levels of soluble CD44 were measured in advanced SM compared to ISM, cutaneous mastocytosis (CM), or healthy controls (p<0.05). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT WT, ROSAKIT D816V, MCPV-1.1, MCPV-1.2, MCPV-1.3, and MCPV-1.4 expressed cytoplasmic and cell surface CD44. To study the mechanisms underlying CD44 expression on MC, we applied various targeted drugs. Incubation with the demethylating agents decitabine (0.1-5 µM) or 5-azacytidine (0.1-5 µM) for 96 hours resulted in a dose-dependent upregulation of CD44 surface expression compared to baseline levels (100%) in all MC lines examined (decitabine, 5 µM: 210±50% in HMC-1.1, 294±76% in HMC-1.2, 236±56% in ROSAKIT WT, 210±54% in ROSAKIT D816V, 242±47% in MCPV-1.1, 179±23% in MCPV-1.2, 207±17% in MCPV-1.3, and 152±5% in MCPV-1.4 cells, p<0.05; 5-azacytidine, 5 µM: 355±104% in HMC-1.1, 429±105% in HMC-1.2, 412±135% in ROSAKIT WT, 292±136% in ROSAKIT D816V, 365±55% in MCPV-1.1, 345±106% in MCPV-1.2, 325±87% in MCPV-1.3, and 278±38% in MCPV-1.4 cells, p<0.05). In contrast, incubation with the MEK-inhibitor refametinib (RDEA119) (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a dose-dependent downregulation of CD44 surface expression compared to baseline levels (100%) in all MC lines examined (refametinib, 2.5 µM: 71±9% in HMC-1.1, 82±3% in HMC-1.2, 33±13% in ROSAKIT WT, 31±3% in ROSAKIT D816V, 62±6% in MCPV-1.1, 82±13% in MCPV-1.2, 84±5% in MCPV-1.3, and 87±1% in MCPV-1.4 cells, p<0.05; pimozide, 7.5 µM: 59±7% HMC-1.1, 68±3% in HMC-1.2, 62±16% in ROSAKIT WT, and 80±3% in ROSAKIT D816V, 62±5% in MCPV-1.1, 72±4% in MCPV-1.2, 73±4% in MCPV-1.3, and 63±9% in MCPV-1.4 cells, p<0.05). These data suggest that the RAS/MEK pathway and the STAT5 pathway are involved in expression of CD44 in neoplastic MC. In order to confirm this hypothesis we transduced HMC-1.2 cells and ROSAKIT WT cells with KRASWT or oncogenic KRASG12V. In all transduced cell lines, KRAS overexpression resulted in upregulation of CD44 surface expression compared to empty vector (100%) (HMC-1.2: 143±17% with KRASWT, 249±53% with KRASG12V, p<0.05; ROSAKIT WT: 170±13% with KRASWT, 403±64% with KRASG12V, p<0.05). As expected, refametinib was found to suppress RAS-induced CD44 expression in these cells. To study the functional role of CD44 in neoplastic MC, we employed a mouse xenotransplantation model using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and shRNA against CD44. In this model, the shRNA-induced knock-down of CD44 in HMC-1.2 cells resulted in reduced MC expansion, reduced tumor formation, delayed ulceration, and prolonged survival compared to cells transduced with control shRNA (median survival in the CD44 shRNA group: 110 days vs 97 days in the control group, p<0.05). The formation of lung metastasis, quantified by human Alu-sequence-specific qPCR, was found to be particularly decreased (15-fold) in the CD44 knock-down group compared to control mice.In conclusion, CD44 is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in patients with advanced SM. Our data also suggest that CD44 is an important mediator of evolution and of malignant spread of neoplastic MC into various organs in SM. Future studies will show whether CD44 can serve as a therapeutic target in patients with advanced SM. DisclosuresHoermann:Gilead: Research Funding; Novartis: Honoraria; Amgen: Honoraria; Ariad: Honoraria. Sperr:Novartis: Honoraria; Amgen: Honoraria, Research Funding. Arock:Agensys, Inc: Research Funding. Valent:Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding.

Prognostic Factors and Survival Prediction in 1,088 Patients with Mastocytosis Collected in the Registry of the European Competence Network on Mastocytosis (ECNM Registry)

Mastocytosis is a hematopoietic disorder characterized by abnormal growth and accumulation of neoplastic mast cells (MC) in various organ systems. Using updated WHO criteria and the proposal of the consensus group, the disease can be divided into cutaneous mastocytosis (CM), indolent systemic mastocytosis (ISM), smouldering SM (SSM), SM with an associated hematologic neoplasm (SM-AHN), aggressive SM (ASM), and mast cell leukemia (MCL). In adult patients with skin involvement but unknown/unavailable bone marrow (BM) studies, the provisional diagnosis ´mastocytosis in the skin (MIS)´ is appropriate. Although this classification has been validated repeatedly and is of prognostic significance, additional prognostic parameters have been identified in recent years (yrs). We have established a patient-registry in the ECNM where over 1,000 cases with confirmed mastocytosis are included. The aim of this study was to identify and validate new prognostic variables predicting survival in patients with mastocytosis and to prepare a simple prognostic scoring system applicable in daily practice. Using the data set of the ECNM registry we analyzed overall survival (OS) and event-free survival (EFS; i.e. until death or progression) in 1,088 patients with mastocytosis (median age: 45.7 yrs; range: 0.1-83.3 yrs, f:m ratio, 1:0.79), including CM (n=152), MIS (n=126), ISM (n=650), SSM (n=26), SM-AHN (n=89), ASM (n=35), and MCL (n=10). The median observation period was 3.5 yrs (75-25% percentile: 1.5-6.9 yrs, maximum 34.1 yrs). In the entire cohort, the median OS was not reached. The probability to be alive after 5, 10, and 20 yrs was 89%, 83%, and 70%, respectively. As expected, the WHO classification turned out to be of utmost predictive significance (Figure 1A; p<0.005). In patients with non-advanced disease, namely CM, MIS, ISM, and SSM, the median survival was not reached, and the survival at 5 yrs was 100%, 97%, 98%, and 85%, respectively, whereas in advanced SM, namely SM-AHN, ASM, and MCL, the median survival was 2.8, 4.1, and 0.8 yrs, respectively. Patients with advanced SM were found to be older, to have higher serum tryptase- and alkaline phosphatase (aPhos) levels, higher white blood counts (WBC), lower hemoglobin (Hb) and platelet (PLT) counts, and more frequently presented with organomegaly (hepatomegaly, splenomegaly, or lymphadenopathy). Moreover, the male/female ratio was higher in advanced mastocytosis. To define the relative impact of the identified risk factors we randomly divided the total cohort (50:50) into a learning set and a validation set, performed uni- and multivariate analyses, and subsequently calculated cut off values for optimal prognostication. In these studies, the poor prognosis of patients with advanced SM was confirmed. In patients with non-advanced disease, all variables tested were significant concerning OS in univariate analyses. However, WBC, Hb, and lactate dehydrogenase had to be excluded due to variance inflation. In multivariate analyses, age >70 yrs, PLT <80 G/L (not related to SM), and aPhos ≥240 U/L were significant predictors concerning OS. Based on these parameters, we established a simple prognostic scoring system. In this score, patients with non-advanced disease (CM, MIS, ISM, SSM) without additional risk factors comprised the low risk group, those with non-advanced disease and presence of one or more risk factors (age >70 yrs, PLT <80 G/L, aPhos ≥240 U/L) the intermediate risk group, and those with advanced disease (with or without additional risk factors) the high risk group. The median OS in the low risk group was not reached, in the intermediate risk group it was 13.5 yrs, and in the high risk group 3.5 yrs (p<0.005; Figure 1B). Significant differences were also observed regarding EFS (p<0.005, Figure 1C). In conclusion, the WHO classification remains the gold-standard of prognostication in patients with mastocytosis, but additional factors, namely age, PLT, and aPhos, are powerful additional variables predicting OS in these patients. Based on in-depth analyses of the ECNM registry data-set, a simple prognostic scoring system for mastocytosis was established and is recommended to define the probability of OS and EFS in patients with mastocytosis in daily practice. [Display omitted] DisclosuresSperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Gotlib:Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Valent:Amgen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Ariad: Honoraria, Research Funding.

Ponatinib Exerts Multiple Effects on Vascular Endothelial Cells: Possible Mechanisms and Explanations for the Adverse Vascular Events Seen in CML Patients Treated with Ponatinib

The potent BCR-ABL1-targeting tyrosine kinase inhibitor (TKI) ponatinib is used for the treatment of patients with drug-resistant chronic myeloid leukemia (CML). However, an increased risk of development of cardiovascular events has been described in CML patients treated with ponatinib. The etiology of these adverse events is currently unknown. In an attempt to discover mechanisms underlying ponatinib-induced adverse vascular events, we have evaluated the effects of ponatinib in vitro on human vascular endothelial cells and on contraction of explanted mice aortic rings. In addition, we examined the effects of ponatinib on angiogenesis in vivo in a mouse model of hind limb ischemia. Ponatinib dose-dependently induced apoptosis in human coronary artery endothelial cells (HCAEC) in a caspase assay (relative apoptosis vs. 1% DMSO: ponatinib 50 nM: 1.79±0.38, p<0.001; ponatinib 100 nM: 2.13±0.42, p<0.001) and this drug effect could partially be blocked by addition of insulin (ponatinib 100 nM + insulin 5 µg/ml: 1.70±0.18, p<0.05). In addition, ponatinib was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) and the human microvascular endothelial cell line HMEC-1, with IC50 values ranging between 100 and 250 nM (p<0.05) as determined by thymidine-incorporation assay. Using a phospho-receptor tyrosine kinase assay in HCAEC, we found that ponatinib also inhibits fetal bovine serum-induced phosphorylation of the VEGF receptor KDR as well as phosphorylation of MER and insulin receptors, which play a role in angiogenesis, vascular homeostasis, and vessel protection. We also found that ponatinib (1 µM, 4 hours) increases adhesion of HUVEC to a plastic-surface compared to DMSO control (Figure). Based on clinical observations of vasoconstriction in ponatinib-treated patients, we also applied ponatinib on aortic rings harvested from C57BL/6 mice. Ponatinib (100 nM, overnight) enhanced norepinephrine-induced vasoconstriction (log EC50: control -7.76±0.06 vs. ponatinib -7.96±0.05, p<0.05, n=6) and inhibited acetylcholine-mediated vasodilatation (log IC50: control -7.45±0.05 vs. ponatinib -7.06±0.1, p<0.001) as shown by myography. These drug effects were blocked by inhibition of nitric oxide (using nitric oxide synthase inhibitor L-NNA, 100 µM) or COX (by applying diclofenac, 3 mg/l), suggesting that ponatinib promotes the generation of vasoconstricting prostanoids. Ponatinib effects were also blocked by the calcium channel blocker nifedipine (1 µM). In C57BL/6 mice, ponatinib (5 mg/kg/day for 35 days) was found to inhibit blood flow recovery in a hind limb ischemia model as shown by Laser-Doppler perfusion imaging after femoral artery ligation. The blood perfusion ratios of the ischemic limb vs. non-ischemic limb at week 5 were: control group: 0.67±0.07 vs. ponatinib: 0.56±0.1; p<0.05). Ponatinib-treated mice also developed toe and foot necrosis more frequently than control mice (necrosis score: control: 0.3 vs. ponatinib: 1.3). In summary, ponatinib affects endothelial cell growth and vasomotor function in-vitro as well as blood flow recovery in a mouse model. These findings might help explain the occurrence of vascular events in CML patients treated with ponatinib and may lead to development of therapeutic strategies for prevention and treatment of ponatinib-induced adverse events. [Display omitted] DisclosuresKirchmair:Ariad: Research Funding. Valent:Ariad: Honoraria, Research Funding; Amgen: Honoraria; Deciphera Pharmaceuticals: Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Abstract A53: The specific FMS kinase inhibitor, DCC-3014, durably inhibits FMS kinase in vivo and blocks cancer bone invasiveness

Abstract Within the tumor microenvironment, tumor-associated macrophages (TAMs) rely on signaling through FMS kinase to promote tumor growth, angiogenesis, vasculogenesis, and metastasis. In addition, FMS-expressing macrophages communicate with the adaptive immune system, which can lead to cancer immunotolerance. Osteoclasts also depend on FMS kinase for differentiation and growth, and play a direct role in the ability of a tumor to metastasize to the bone and progress. Taken together, FMS kinase is an excellent target for small molecule therapy of tumor growth and bone metastasis. Using Deciphera Pharmaceuticals' approach to kinase inhibition, potent and specific FMS kinase inhibitors have been identified that achieve single digit nanomolar inhibition in biochemical and cellular assays of FMS activity. Due to their binding mode, these inhibitors retain potency in the presence of high ATP concentrations and also exhibit long off-rates from FMS kinase. Development candidate DCC-3014 is highly selective, inhibiting no other kinases within 100-fold of FMS potency, including related kinases KIT, PDGFRs, FLT3, and VEGFR2. DCC-3014 exhibits potent and durable inhibition of FMS in vivo in a pharmacokinetic/pharmacodynamic model. In a variety of cancer models, DCC-3014 or related analogs slow tumor growth, decrease tumor-promoting macrophages, and protect against osteolytic bone invasion. DCC-3014 is currently undergoing formal preclinical development. Citation Format: Bryan D. Smith, Michael D. Kaufman, Cynthia B. Leary, Molly M. Hood, Wei-Ping Lu, Benjamin A. Turner, Subha Vogeti, Scott C. Wise, Daniel L. Flynn. The specific FMS kinase inhibitor, DCC-3014, durably inhibits FMS kinase in vivo and blocks cancer bone invasiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A53. doi:10.1158/1538-7445.CHTME14-A53

AI-25 * THE NOVEL c-MET INHIBITOR ALTIRATINIB (DCC-2701) INHIBITS TUMOR GROWTH AND INVASION IN A BEVACIZUMAB RESISTANT GLIOBLASTOMA MOUSE MODEL

Glioblastoma with acquired resistance to bevacizumab therapy overexpresses c-MET which mediates tumor growth, EMT, invasion, and metastasis. We evaluated the expression level of total and phosphorylated c-MET (p-c-MET) and the inhibitory activity of altiratinib (DCC-2701), a novel c-MET/TIE2/VEGFR inhibitor (Deciphera Pharmaceuticals) that has demonstrated significant BBB penetration into the CNS. Four of 12 (30%) mesenchymal glioma stem cell lines (GSCs) highly expressed c-Met and were sensitive to altiratinib treatment in vitro for blocking cell viability. After HGF stimulation, the expression level of p-cMET was significantly increased in these 4 cell lines. Altiratinib markedly suppressed HGF stimulated p-c-MET expression in both paracrine (MET+) and autocrine (MET + /HGF+) GSC cell lines. In an intracranial glioma mouse model, animals were treated with bevacizumab (Bev) alone, altiratinib alone or the combination. Mice were euthanized at 3, 4 and 5 weeks. H&E staining revealed a significant reduction in tumor size in the combination group compared with the tumors treated with either agent alone. Altiratinib alone or combination with Bev treatment dramatically suppressed EMT and tumor invasion in vivo. This anti-invasive effect correlated with decreased N-cadherin and vimentin levels. In vivo, microvascular density was significantly inhibited in the groups treated with altiratinib and altiratinib plus Bev compared with control or the group treated with Bev alone. Similarly, F4/80+ BMDC cell infiltration was significantly suppressed in the groups treated with altiratinib and altiratinib plus Bev combination compared to control Bev-treated groups. In summary, our data indicated that altiratinib inhibited tumor growth, EMT and invasion both in vitro and in vivo. The combination of altiratinib and anti-VEGF therapy may provide a new strategy to overcome Bev resistance and prolong overall survival in patients with glioblastoma.