Number Of Tyrosine Kinase Inhibitors Research Articles

Targeting the FLT3 Mutation in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) exhibiting an internal tandem duplication of the FLT3 gene (FLT3-ITD) is an aggressive haematologic malignancy with a poor prognosis due to a high relapse rate and very limited options after relapse with conventional salvage regimens, whereas the prognostic impact of point mutations in the tyrosine kinase domain of the FLT3 gene (FLT3-TKD) are less clear. A number of tyrosine kinase inhibitors (TKIs) have been developed that inhibit the constitutively activated kinase activity caused by the FLT3 mutation, thus interrupting signalling pathways. Early clinical trials of these agents as monotherapy failed to elicit enduring complete responses, leading to clinical testing of FLT3 TKI in combination with conventional chemotherapy. Midostaurin has demonstrated improved survival in combination with standard intensive chemotherapy as compared to standard chemotherapy alone in younger adult patients with newly diagnosed FLT3-mutated AML and is the first and currently the only approved FLT3 TKI. Newer, more selective compounds, such as gilteritinib and crenolanib, have also demonstrated significant potency and specificity. Several combination trials are ongoing or planned in both relapsed and newly diagnosed AML patients with activating FLT3 mutations.

Exploring Pre-B Cell Receptor Signalling As a Therapeutic Target in Acute Lymphoblastic Leukaemia

Introduction:Acute lymphoblastic leukaemia (ALL) is a clonal disorder of developing lymphocytes and is the most common malignancy in children and adolescents. While cure rates are high, treatment is associated with significant morbidity and relapsed ALL remains one of the leading causes of cancer-related deaths in children. New, less toxic therapies are clearly needed for refractory ALL.There are a number of lines of evidence to suggest that ALL cells hijack components of precursor-B cell receptor (Pre-BCR) signalling and this dependency may be amenable to therapeutic exploitation. There are a number of tyrosine kinase inhibitors (TKIs) targeting Pre-BCR signalling that are showing great promise in the clinic for other leukemia subtypes which warrant preclinical evaluation in childhood ALL.[1] These include CAL-101 (PI3K-δ inhibitor), Ibrutinib (BTK inhibitor), Fostamatinib R406 (SYK inhibitor) and Dasatinib (BCR-ABL/SRC inhibitor).Methods:TKIs were evaluated in ALL cells, including cell lines (PreB 697 and its glucocorticoid resistant descendant, R3F9; Nalm-6 and Reh) and 25 patient derived xenograft samples (PDX) from 12 predominantly high risk/relapse children ALLs. Resazurin was used to assess cell viability. Flow cytometry was used to detect Pre-BCR expression (µHc, Vpreb and λ5) and functionality using a Calcium flux assay. Phospho-flow cytometry was performed to monitor constitutive phosphorylation and response to Pre-BCR activation and to assess pharmacodynamic drug action (p-AKT, p-BLNK, p-BTK, p-SYK, p-ERK, and p-PLC-ϒ2). GILZ expression was measured by RQ-PCR. Cell cycle and apoptosis were determined by flow cytometry using Propidium Iodide and Annexin V staining.Results:ALL cell lines were resistant to CAL-101 (mean GI50 52.08 µM, range 25 µM-77.83 µM) and Ibrutinib (mean GI50 15.9 µM, range 11.47 µM-18.3 µM). However, modest sensitivity was seen to R406 (mean GI50 4.32 µM, range 2.88 µM-5.83 µM) and Dasatinib (mean GI50 5.33 µM, range 2.45 µM-12.5 µM). CAL-101 and Dasatinib were shown to be cytostatic, causing G1 arrest but no significant apoptosis, while Ibrutinib and R406 were associated with cell cycle arrest and significant apoptosis after 72 hours incubation with GI50 concentrations (16.81±1.71 % and 31.34±5.78 % apoptosis, respectively). Pre-B receptor positive cells were more sensitive to Dasatinib and Fostamatinib. Pharmacodynamic assessment using Phospho-flow cytometry and Western blotting showed inhibition of the relevant targets at the GI50 concentrations. PDX ALL cells were generally more sensitive than the cell lines; CAL-101 (mean GI50 25.56 µM, range 76 nM-100 µM; 2 out of 12 patient samples <2µM); Ibrutinib (mean GI50 14.23 µM, range 490 nM-100 µM; 3 out of 12 patient samples <5µM); R406 (mean GI50 11.52 µM and range 56 nM-25 µM, 4 out of 12 patient samples <4µM); Dasatinib (mean GI50 25.56 µM, range 76 nM-100 µM; 3 out of 12 patient samples <0.5µM). ALLs sensitive to Dasatinib were Ph+ or Pre-BCR positive and the latter were also sensitive to Fostamatinib.Synergism was seen after co-treatment of TKIs with the glucocorticoid (GC), Dexamethasone (CAL-101 CI mean 0.7, range 0.056-1.34; Ibrutinib CI mean 0.71, range 0.41-0.97; R406 CI mean 0.27, range 0.1-0.6 and Dasatinib CI mean 0.63, range 0.14-1.29). No synergism was observed in the glucocorticoid receptor negative, Reh cell line. For R406 and Dasatinib, co- exposure was strongly synergistic and was associated with increased apoptosis in PreB 697 and the GC resistant lines, Nalm-6 and R3F9. Synergism was associated with a significant increase in expression of the GR target, GILZ and an enhanced downregulation of R406 and Dasatinib targets (p-SYK for R406 and p-BTK, p-SYK for Dasatinib).Conclusion:We have identified significant sensitivity of TKIs impacting on Pre-BCR signalling in ALL cells at clinically relevant concentrations; Pre-BCR positive ALLs were associated with Dasatinib and Fostamatinib sensitivity; Pre-BCR negative ALL cells were also sensitive to some TKIs, although predictive biomarkers remain to be established. Marked synergism was observed in combination with dexamethasone, even in GC resistant cells. In vivo preclinical confirmation of these data may offer new therapies for refractory ALL.1. Young, R.M. and L.M. Staudt, Targeting pathological B cell receptor signalling in lymphoid malignancies. Nature Reviews Drug Discovery, 2013. 12(3): p. 229-243. DisclosuresNo relevant conflicts of interest to declare.

Risk of hepatotoxicity with concurrent statin and tyrosine kinase inhibitor (TKI) or mTOR inhibitor (mTORi) in patients with metastatic renal cell carcinoma (mRCC).

610 Background: TKIs and statins alone can cause liver injury. When used together, there is additive risk for hepatotoxicity, as seen with pazopanib and simvastatin, and the potential for discontinuing TKI therapy. Liver enzyme abnormalities are also associated with mTORi. Hepatotoxicity from concurrent use of statins as a class with TKI or mTORi as a class in real world practice in mRCC has not been reported. Methods: This observational cohort study included adult patients treated with a TKI or mTORi for mRCC at the Huntsman Cancer Institute from 2004-2014. We performed a treatment line analysis. For each treatment line, CTCAE criteria were used to categorize maximum AST, ALT, and total bilirubin. Highest grade of any liver enzyme was compared between TKI with and without statin and between mTORi with and without statin (Fisher’s Exact test). Results: A total of 162 treatment lines were included. For TKI and statin, the three most common treatment line combinations were sunitinib and simvastatin (7), sunitinib and atorvastatin (4), and pazopanib and simvastatin (3). For mTORi and statin, the three most common treatment line combinations involved simvastatin with temsirolimus (7) and everolimus (3). Prevalence of hepatotoxicity is shown in the table. Conclusions: Our results show a trend toward significance in the prevalence of hepatotoxicity with concomitant mTORi and statin versus mTORi alone, perhaps explained by mTORi-mediated competitive inhibition of simvastatin (sensitive CYP 3A4 substrate). There was no difference in hepatotoxicity for TKI with or without statin, although there were few pazopanib and simvastatin combinations. Study limitations include the small sample size, limited number of TKI or mTORi and statin combinations, and retrospective study design. A larger study is needed to validate these findings. [Table: see text]

Russian Registry of Chronic Myeloid Leukemia Management in Routine Clinical Practice-Local Therapy and Monitoring

▪Introduction: in the last 10 years the number of tyrosine kinase inhibitors (TKI) used to treat patients with chronic myeloid leukemia (CML) significantly increased. The basic principle of therapy was also changed, now it is aimed at early induction of deep response (ELN, NCCN). The treatment results in clinical practice may vary widely and depend on the effectiveness of the entire complex TKI, based on timely evaluation of residual leukemic clone.Objective: To characterize the TKI treatment results and monitoring of residual leukemic clone in clinical practice in CML patients in the Russian Federation (RF).Methods: The observational study "Russian registry of chronic myeloid leukemia management in routine clinical practice" (CSTI571ARU06 LLC) was launched in November 2011 by Novartis. Patients with Ph'+ CML were enrolled into study after signing the informed consent. Thus the study has a retrospective part (4626 patients) and prospective group of 1828 CML patients. By February 2015, the Registry contained information about 6466 patients from 110 centers of 80 regions of the RF. The annual increase of newly registered CML cases ranged from 493 to 694 people per year or 10-13% of the total number of patients included in the Registry on the treatment of CML in 2012- 2014. Characteristics of patients in the Registry: median duration of the disease was 71 months (from 0.2 to 343 months); median age at registration time and CML diagnosis was 49.5 years (range 2 - 94 years); male 43%. The biggest number of cases was diagnosing with patients aged 50-69 years old, which is 50% out of all the registered patients. It is important to note that the number of female patients aged 50 years old and older (39.1% in total, out of the overall number of CML cases) prevails the same male patients age group (25.6% of the cases). Chronic phase was in 93.6% of patients, accelerated phase and blast crisis- 5.6% and 0.6% cases, accordantly. Sokal risk group's ratio was 50%/31%/19% for low, intermediate and high risk, respectively.Results: The data for the analysis were available for 5632 patients (93% of CML patients in the Registry). The 1st line TKI therapy was Imatinib (IM)- in 4908 of the 5632 patients (87%), including the advanced phases of CML; in 71 and 14 patients the 1st line treatment was nilotinib and dasatinib respectively(totally 1%). The percentage of patients receiving TKI-2 ranged from 10 to 35% in different regions of RF. In 2006, the diagnosis CML was confirmed by the data of karyological analysis only with 42% of the patients, but it should be pointed out that currently cytogenetic and/or molecular-based analysis has been conducted at least once with 99% of the patients. Thus, the data presented in Registry of CML treatment provides grounds to state that almost all the patients have had their diagnosis verified within routine clinical practice. The presented data demonstrate the amount of work done to obtain such results during the previous ten-year period. We evaluated cytogenetic monitoring (CyM)/molecular monitoring (MM) frequency during years 2013-2014. Two or more studies per year of cytogenetic CyM/MM were done only 20% of 40 patients per year (2320 persons); with 50% (2960 in 2013, 1203 in 2014) of patients CyM/MM was performed once; no studies have been done in 489 patients in 2013 and 3580 patients in 2014. There was a trend of increasing of molecular studies to evaluate minimal residual disease:1829 MM vs 491 CyM in 2013 and 1028 MM vs 179 CyM in 2014. The absence of optimal response (ELN2013) was observed in 887 patients on different TKIs: (759patients on IM: in 732 patients with the duration of IM therapy over 12months and in 27 patients-less than 12months. Change of treatment to 2nd line was performed in 639 (12%) patients. The death occurred in 373 (6.2%) patients, no data of the living status was in 50 (0.8%) cases. According to the Registry data, death due to the CML progression was in 99 cases (26%).Conclusions: The observational study "Russian registry of chronic myeloid leukemia management in routine clinical practice" is the largest registry of hematological diseases in Russia, providing information about CML patients receiving TKI therapy more than 10 years. The introduction of information technologies to analyze a large amount of data is an essential component to improve the quality of medical care. The evaluation of therapy results can give objective information of the TKI 1st and 2nd generations requirement. DisclosuresTurkina:Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis Pharma: Consultancy. Golenkov:Novartis: Consultancy.

Survival Impact of Patients (Pts) with Chronic Myeloid Leukemia (CML) Due to Failure from the Use of One or More Tyrosine Kinase Inhibitors (TKI)

▪Background: Imatinib induces durable complete cytogenetic remissions (CCyR) in at least two thirds of pts with chronic phase (CP) CML, most of them also achieving a major molecular response (MMR). However, some pts become intolerant or resistant to imatinib and require change of therapy to a 2nd or 3rd generation TKIs (dasatinib, nilotinib, bosutinib, and/or ponatinib). The projected 3-year survival for these pts was reported as 72% (Kantarjian et al. Cancer 2007) at a time when treatment options for such pts was limited. A smaller subset of pts may require additional changes to a third or fourth TKI. The impact of sequential TKI therapy, although standard practice, has not been well studied. We analyzed the long term outcome of pts receiving multiple TKIs.Method: We analyzed the medical records of 1775 pts with CML-CP treated at a single institution between 02/2000 and 07/2015. Among them 582 (33%) received more than one TKI as follows: 2TKIs (n=370), 3TKIs (n=130), and 4+TKIs (n=82; 4 TKI n=59, 5 TKI n=20, 6 TKI n=1, 7 TKI n=2). For the purpose of this analysis, a TKI used more than once was counted as 2 TKI provided there was a different TKI used between the two periods when the TKI in question was used. We analyzed pt's characteristics, response to therapy, transformation to accelerated and blast phase, and long term outcomes. Overall Survival (OS) and Transformation-Free Survival (TFS) probabilities were measured using Kaplan-Meier method starting from the date they began to use the their 2nd TKI and grouped by the number of TKI used.Results: The number of CML pts in early CP (diagnosis to start of therapy, ≤ 12 months) that used 2, 3, and 4+TKIs was 229, 54, and 34, respectively. The number of pts in late CP (diagnosis to start of therapy >12 months) that used 2, 3, and 4+TKIs was 141, 76 and 48 respectively. The median age (range) for pts that used 2, 3, and 4+ TKIs were 48 yrs (15-81), 52 yrs (18-87), and 53 yrs (18-80), respectively. The ratio of males to females in each cohort was 187:183, 58:72, and 37:45, respectively. The median time from diagnosis to 2nd, 3rd and 4th TKI was 2, 4, and 6 years respectively. For the 3 cohorts (2, 3, 4+TKIs), TKIs used included imatinib (n=322, n=130, n=79), dasatinib (n=227, n=112, n=77), nilotinib (n=119, n=109, n=70), ponatinib (n=44, n=27, n=37) and bosutinib (n=26, n=13, n=29), respectively. The 3-year overall survival probability for pts treated with 2 TKI was 88% (median survival not reached); for those that used 3 TKI 72% (median survival not reached), and for those that used 4+ TKI 48% (median survival 34 months) (Fig. 1). Corresponding 5-year figures are 80%, 53% and 38%. The 3-year transformation-free survival probability for pts treated with 2 TKI was 93%; for those treated with 3 TKI 88%; and for pts treated with 4 TKI 80% (median not reached in any cohort ) (Fig. 2). Corresponding figures for 5-years are 90%, 84%, and 75%.Conclusion: With more and improved treatment options, pts with resistance or intolerance to multiple TKI have the potential for long-term survival. Nearly 80% of pts who receive 2 TKI are alive at 5 years and more than 90% remain in chronic phase. As pts experience subsequent failure, the outcome worsens. These results provide information that may help with treatment planning and set the expectations against which treatment options used in such pts should be measured. [Display omitted] [Display omitted] DisclosuresWierda:Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Daver:ImmunoGen: Other: clinical trial, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Cortes:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; BMS: Consultancy, Research Funding; Teva: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

A systematic analysis of the resistance and sensitivity of HER2YVMA receptor tyrosine kinase mutant to tyrosine kinase inhibitors in HER2-positive lung cancer

Human epidermal growth factor receptor 2 (HER2) has become a well-established target for the treatment of HER2-positive lung cancer. However, a frequently observed in-frame mutation that inserts amino acid quadruplex Tyr776-Val777-Met778-Ala779 at G776 (G776YVMA) in HER2 kinase domain can cause drug resistance and sensitivity, largely limiting the application of reversible tyrosine kinase inhibitors in lung cancer therapy. A systematic investigation of the intermolecular interactions between the HER2YVMA mutant and clinical small-molecule inhibitors would help to establish a complete picture of drug response to HER2 G776YVMA insertion in lung cancer, and to design new tyrosine kinase inhibitors with high potency and selectivity to target the lung cancer-related HER2YVMA mutant. Here, we combined homology modeling, ligand grafting, structure minimization, molecular simulation and binding affinity analysis to profile a number of tyrosine kinase inhibitors against the G776YVMA insertion in HER2. It is found that the insertion is far away from HER2 active pocket and thus cannot contact inhibitor ligand directly. However, the insertion is expected to induce marked allosteric effect on some regions around the pocket, including A-loop and hinges connecting between the N- and C-lobes of HER2 kinase domain, which may exert indirect influence to inhibitor binding. Most investigated inhibitors exhibit weak binding strength to both wild-type and mutant HER2, which can be attributed to steric hindrance that impairs ligand compatibility with HER2 active pocket. However, the cognate inhibitor lapatinib and the non-cognate inhibitor bosutinib were predicted to have low affinity for wild-type HER2 but high affinity for HER2YVMA mutant, which was confirmed by subsequent kinase assay experiments; the inhibitory potencies of bosutinib against wild-type and mutant HER2 were determined to be IC50 > 1000 and =27 nM, respectively, suggesting that the bosutinib might be exploited as a selective inhibitor for mutant over wild-type HER2. Structural examination revealed that formation of additional non-bonded interactions such as hydrogen bonds and hydrophobic contacts with HER2 A-loop region due to G776YVMA insertion is the primary factor to improve bosutinib affinity upon the mutation.

Перспективы терапевтического воздействия на сигнальный путь FGFR

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in key cellular functions and cancerogenesis. Nowadays FGFR and their ligands are one of the most investigated markers in oncology and targets for specific therapy. There are a lot of clinical trials in on- cology include drugs with anti-FGFR activities. The most of these drugs are tyrosine kinase inhibitors and monoclonal antibodies. When we say about therapeutic effects on the FGFR signaling pathway, we say about opportunity not only block the ligands and FGFRs, but the under- lying molecular and signaling pathways activated by FGFRs. Nowadays the number of tyrosine kinase inhibitors selectively blocking FGFRs is extremely small. Typically, tyrosine kinase inhibitors can block a wide range of targets. Some of these inhibitors have entered in clinical practice in the treatment of metastatic tumors of different localizations, others are in clinical trials. On August 2014, 74 studies investigating inhibitors of FGFRs are registered on clinicaltrials.gov. A number of marketed drugs at high concentrations also has the ability to inhibit FGFR – sorafenib, vandetanib, motesanib, however, increasing the concentration of these drugs is associated with severe toxicity of treat- ment. In the recommended therapeutic concentrations, adequate blocking FGFR tyrosine kinase domain is doubtful. The review paid atten- tion to such drugs as pazopanib, nintedanib, cediranib, brivanib, dovitinib, ponatinib. We showed the results of treatment with inhibitors of FGFR in different cancers such as breast cancer, colon cancer, endometrial cancer, gastric cancer, thyroid cancer, lung cancer, ovarian cancer. Despite the fact that anti-FGFR therapy are at an early stage of clinical investigation, some difficulties in implementing this thera- peutic approach have been seen, such as high toxicity, not validated targets, the need for patient selection, depending on the activity of FGF– FGFR pathway, as well as mutations in genes of downstream molecular signaling pathways. In summary in the article we reviewed relevant literature to identify current status, difficulties and future perspectives in development of anti- FGFR drugs. In this article, we review FGFR signaling and describe the therapeutic intervention in patients with solid tumors.

Immune consequences of tyrosine kinase inhibitors that synergize with cancer immunotherapy.

Combination therapy for the treatment of cancer is becoming increasingly essential as we gain improved understanding of the complexity of cancer progression and the mechanisms by which cancer cells become resistant to single-agent therapy. Recent studies, both clinical and preclinical, have suggested that immunotherapy is a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still needed. A number of recent studies have indicated that antiangiogenic tyrosine kinase inhibitors (TKIs) target multiple components of the tumor microenvironment and are an ideal class of agents for synergizing with cancer immunotherapy. TKIs are well known to modulate tumor endothelial cells, leading to vascular normalization; however, these agents have also been recently shown to decrease tumor compactness and tight junctions, thereby reducing solid tumor pressure and allowing for improved perfusion of collapsed vessels and increased tumor oxygenation. In addition, some TKIs are capable of inducing immunogenic modulation, whereby tumor cells are sensitized to killing by T lymphocytes. Moreover, a number of TKIs have been shown to be involved in immune subset conditioning, increasing the frequency and function of effector immune elements, while decreasing the number and function of immune suppressor cells. The alteration of the immune landscape, direct modification of tumor cells, and improved vascular perfusion leads to improved antitumor efficacy when antiangiogenic TKIs are combined with immunotherapy. Collectively, the data presented in this review support the clinical combination of multi-targeted antiangiogenic TKIs, including but not limited to cabozantinib, sunitinib, and sorafenib, as well as to other antiangiogenic therapies, such as the anti-VEGF antibody bevacizumab, with cancer vaccines for improved treatment of solid tumors.

Abstract 3083: The genetic landscape of Ph-like acute lymphoblastic leukemia

Abstract BCR-ABL1-like, or “Ph-like” B-progenitor acute lymphoblastic leukemia (B-ALL) constitutes up to 15% of childhood and 30% of adult ALL, and is characterized by a gene expression profile similar to BCR-ABL1 ALL, alteration of IKZF1, and poor outcome. A pilot next-generation sequencing study identified kinase activating alterations in 15 Ph-like ALL cases. The goals of this study were to define the genomic landscape of Ph-like ALL in children and young adults, and to examine the utility of tyrosine kinase inhibitors (TKIs) in patients harboring genetic alterations activating kinase signaling. We studied 1665 B-ALL cases, including 309 childhood standard risk (10.8% Ph-like), 826 childhood high risk (14% Ph-like), 370 adolescent (16-21 years, 21% Ph-like) and 160 young adult (21-39 years; 26% Ph-like) cases. Approximately 50% of Ph-like cases harbored a CRLF2 rearrangement (IGH-CRLF2 or P2RY8-CRLF2). Next-generation sequencing was performed for 160 non-CRLF2 expressing Ph-like cases, including mRNA-seq (141 cases), whole genome sequencing (30 cases) and/or exome sequencing (12 cases). Fusion transcripts were identified using CICERO, a novel mRNA-seq assembly-based structural variation detection method. Over 100 chimeric in-frame fusions were identified, including 29 involving 12 tyrosine kinase or cytokine receptor genes, 15 of which were recurrent: JAK2 (10 partners), ABL1 (6), ABL2 (3), PDGFRB (3), CSF1R, TYK2, NTRK3, PTK2B, IL2RB (1 partner each), and rearrangements of EPOR into the IGH and IGK loci. Together, these rearrangements were present in ∼30% of Ph-like ALL cases. Additional sequence and structural alterations activating kinase signaling were identified in ∼10% of cases (e.g IL7R, FLT3, SH2B3). Despite the diversity of kinase alterations, the majority are predicted to respond to a limited number of TKIs, but many are novel or have not been tested in suitable preclinical models of ALL. We show that expression of RCSD1-ABL1, RANBP2-ABL1, ZMIZ1-ABL1, RCSD1-ABL2, SSBP2-CFS1R and PAX5-JAK2 in Ba/F3 and primary mouse pre-B cultures induces cytokine-independent proliferation and constitutive activation of JAK/STAT signaling. Furthermore, the ABL1, ABL2 and CSF1R fusions were sensitive to dasatinib (IC50 range 1-2nM), whilst PAX5-JAK2 only responded to the JAK2 inhibitor, ruxolitinib. Notably, we show efficacy of dasatinib (20mg/kg/day p.o) in a xenograft model of ETV6-ABL1, with reduction of circulating human CD45+ cells (17.4 vs 88.2%; p&amp;lt;0.0001) and spleen weight (117 vs 321mg; p&amp;lt;0.0001) in dasatinib treated mice (n=5) compared to vehicle treated mice (n=5). These data define the genomic landscape of Ph-like ALL and show that the majority of cases harbor genetic alterations that activate a limited number of kinase signaling pathways. These results provide the basis for prospective precision medicine clinical trials that identify and direct patients with Ph-like ALL to logical TKI therapy. Citation Format: Kathryn G. Roberts, Yongjin Li, Debbie Payne-Turner, Jinghui Zhang, Richard C. Harvey, Yung-Li Yang, Guangchun Song, Jing Ma, Shann-Ching Chen, Jinjun Cheng, Natalia Santiago-Morales, Ilaria Iacobucci, Meenakshi Devidas, I-Ming Chen, Shalini Reshmi, Michael Rusch, Pankaj Gupta, Naomi J. Winick, William L. Carroll, Nyla A. Heerema, Andrew J. Carroll, Elizabeth A. Raetz, Guido Marcucci, Clara D. Bloomfield, Wendy Stock, Steven M. Kornblau, Elisabeth Paietta, Ching-Hon Pui, Sima Jeha, James Downing, Daniela S. Gerhard, Julie M. Gastier-Foster, Mignon L. Loh, Cheryl Willman, Stephen P. Hunger, Charles G. Mullighan. The genetic landscape of Ph-like acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2014-3083

Inhibition of Receptor Signaling and of Glioblastoma-derived Tumor Growth by a Novel PDGFRβ Aptamer

Platelet-derived growth factor receptor β (PDGFRβ) is a cell-surface tyrosine kinase receptor implicated in several cellular processes including proliferation, migration, and angiogenesis. It represents a compelling therapeutic target in many human tumors, including glioma. A number of tyrosine kinase inhibitors under development as antitumor agents have been found to inhibit PDGFRβ. However, they are not selective as they present multiple tyrosine kinase targets. Here, we report a novel PDGFRβ-specific antagonist represented by a nuclease-resistant RNA-aptamer, named Gint4.T. This aptamer is able to specifically bind to the human PDGFRβ ectodomain (Kd: 9.6 nmol/l) causing a strong inhibition of ligand-dependent receptor activation and of downstream signaling in cell lines and primary cultures of human glioblastoma cells. Moreover, Gint4.T aptamer drastically inhibits cell migration and proliferation, induces differentiation, and blocks tumor growth in vivo. In addition, Gint4.T aptamer prevents PDGFRβ heterodimerization with and resultant transactivation of epidermal growth factor receptor. As a result, the combination of Gint4.T and an epidermal growth factor receptor-targeted aptamer is better at slowing tumor growth than either single aptamer alone. These findings reveal Gint4.T as a PDGFRβ-drug candidate with translational potential.

Membrane Tyrosine Kinase Receptors Kit and FLT3 are an Important Targets for the Therapy of Acute Myeloid Leukemia

KIT and FLT3 are class III trans membrane tyrosine kinases playing key roles in the control of hematopoietic stem cell survival and proliferation. KIT is mutated in about 2.5-3% of acute myeloid leukemia (AML) patients mainly by point mutations occurring at the level of tyrosine kinase domains. FLT3 is mutated in about 30% of AML patients, either by internal tandem duplication of the juxtamembrane domain or by point mutations occurring at the level of tyrosine kinase domains. All these types of mutations lead to the constitutive activation of KIT and FLT3 receptors, respectively. In some AML subsets, the occurrence of FLT3/ITD mutation is associated with a poor prognosis. These observations have represented the basis for the development of a large number of Tyrosine Kinase Inhibitors (TKIs) with activity against KIT and FLT3. Some of these inhibitors are still in the preclinical phase, but many have been tested in phase I/II/III clinical studies. Although these inhibitors when used as single agents have lead to a significant reduction of the number of leukemic blasts, they have produced only transient and limited clinical responses. This efficacy may be related to the limited inhibitory caefficacy of these inhibitors, occurrence of resistance and to the complexity of the genetic abnormalities occurring in AMLs. However, recently Quizartinib, a potent FLT3 inhibitor, showed an impressive rate of remission induction in patients with chemotherapy-refractory FLT3-ITD-positive AMLs, but most remissions do not meet classical criteria of complete remissions and instead represent clearance of bone marrow blasts with incomplete recovery of blood cell counts. However, the majority of these patients after an initial sustained response exhibit a leukemic relapse due to the rare pre-exisisting or new FLT3-ITD-TKD mutants surviving to the treatment with this drug and becaming driver leukemic clones. The combination of various types of FLT3 TKIs, including a type II TKI like Quizartinib with a type I FLT3 inhibitor efficiently targeting TKD mutants, such as Crenolanib, or the combination of FLT3 inhibitors with other molecularly targeted agents inhibiting other pathways activated in leukemic cells would determine a major progress in leukemia therapy. The efficacy of FLT3 TKIs in combination with standard anti-leukemic chemotherapeutics is also under evaluation in several combination clinical trials. It is expected that these trials could determine an improvement in the negative prognosis of AML patients with FLT3/ITD mutations. Keywords: Membrane tyrosine kinase receptors, leukemia, cytogenetic.

Lapatinib impact on pulmonary hypertension.

160 Background: Pulmonary Arterial Hypertension (PAH) and cancer share growth factor and protein kinase signaling pathways that result in smooth muscle cell proliferation and vasculopathy. A number of tyrosine kinase inhibitors (TKI) have been developed for the treatment of cancer, and some of these like Imatinib have been investigated in PAH. There is little known about the impact of other TKI on the pulmonary vasculature. After reporting a case of lapatinib-induced PAH that reversed after withdrawal of the drug, we investigated the association of lapatinib with the development of PAH in our institution. Methods: A retrospective chart review was conducted, in accordance with IRB protocol, of all patients treated with lapatinib over the past five years at our institution. Patients who had undergone 2-D echocardiogram both prior to and after treatment were included in the analysis. Systolic Pulmonary Artery Pressures (PASP), right-sided chamber dilation and function, TAPSE were each evaluated. Through chart review, patients were evaluated in terms of risk factors for non-Group 1 PAH. A Wilcoxon signed-rank sum test was used to compare the changes in PASP before and after the initiation of lapatinib. Results: A total of 27 patients were found to have 2-D echo done before and after starting treatment with lapatinib. All patients had normal PASP before starting treatment. Eight patients developed elevated PASP following treatment. Two patients had evidence of left-sided heart disease felt to contribute to their elevated pressures, while the remaining 6 patients had no evidence for Group 2 (associated with left heart disease), 3 (associated with lung disease or hypoxemia) or chronic thromboembolic PAH. Right heart catheterization was available in one patient, confirming the diagnosis of PAH. The median pre-treatment and post treatment PASP in those 6 patients was 29.5 mmHg and 65.5 mmHg respectively (N=6; p = 0.03). Conclusions: Lapatinib is a TKI with a selective inhibition to EGFR and HER2, approved by FDA in 2007 as a second line agent in advanced breast cancer. Given the potential of TKIs as anti-proliferative agents in PAH, a number are currently under study, but little is yet known. This is the first study in the literature to show that PAH is a potential side effect of lapatinib.

Cardiovascular Safety of Tyrosine Kinase Inhibitors: With a Special Focus on Cardiac Repolarisation (QT Interval)

The development of tyrosine kinase inhibitors (TKI) represents a major milestone in oncology. However, their use has been found to be associated with serious toxicities that impinge on various vital organs including the heart. Sixteen TKIs have been approved for use in oncology as of 30 September 2012, and a large number of others are in development or under regulatory review. Cardiovascular safety of medicinal products is a major public health issue that has concerned all the stakeholders. This review focuses on three specific cardiovascular safety aspects of TKIs, namely their propensity to induce QT interval prolongation, left ventricular (LV) dysfunction and hypertension (both systemic and pulmonary). Analyses of information in drug labels, the data submitted to the regulatory authorities and the published literature show that a number of TKIs are associated with these undesirable effects. Whereas LV dysfunction and systemic hypertension are on-target effects related to the inhibition of ligand-related signalling pathways, QT interval prolongation appears to be an off-target class III electrophysiologic effect, possibly related to the presence of a fluorine-based pharmacophore. If not adequately managed, these cardiovascular effects significantly increase the morbidity and mortality in a population already at high risk. Hitherto, the QT effect of most QT-prolonging TKIs (except lapatinib, nilotinib, sunitinib and vandetanib) is relatively mild at clinical doses and has not led to appreciable morbidity clinically. In contrast, LV dysfunction and untreated hypertension have resulted in significant morbidity. Inevitably, dilemmas arise in determining the risk/benefit of a TKI therapy in an individual patient who develops any of these effects following the treatment of the TKI-sensitive cancer. QT interval prolongation, hypertension and LV dysfunction can be managed effectively by using reliable methods of measurement and careful monitoring of patients whose clinical management requires optimisation by a close collaboration between an oncologist and a cardiologist, an evolving subspecialty referred to as cardio-oncology. Despite their potential adverse clinical impact, the effects of TKIs on hypertension and LV function are generally inadequately characterised during their development. As has been the case with QT liability of drugs, there is now a persuasive case for a regulatory requirement to study TKIs systematically for these effects. Furthermore, since most of these novel drugs are studied in trials with relatively small sample sizes and approved on an expedited basis, there is also a compelling case for their effective pharmacovigilance and on-going reassessment of their risk/benefit after approval.

Abstract 3035: Targeting of PDGFRβ with a novel RNA-aptamer inhibits glioma cell survival, growth, invasion and enhances antitumor activity of an anti-EGFR aptamer.

Abstract The platelet-derived growth factor receptor (PDGFR) is an important member of receptor tyrosine kinase (RTK) family. The activation of PDGFRβ signaling pathway induces various cellular responses, including cell proliferation, migration and angiogenesis. Preclinical studies have not only shown a central role for the overexpression and deregulated activation of PDGFRβ-mediated signaling in development of many tumors, including glioma, and the maintenance of the malignant phenotype, but have also demonstrated that the targeted inhibition of signaling cascades has significant anti-cancer effects. Also, several studies have reported that epidermal growth factor receptor (EGFR) cross-talk with PDGFRβ potentially leads to malignant glioblastoma resistance to anti-EGFR therapy and that EGFR transactivation contributes importantly to PDGFRß signal transduction. Overall these data indicate that PDGFRβ represents a valuable target for tumor therapeutic development. A number of tyrosine kinase inhibitors under development as anti-tumor agents have been found to inhibit the PDGFRβ. However, these compounds are not selective and have multiple tyrosine kinase targets. Aptamers are single-stranded oligonucleotides able to bind at high affinity to specific protein by folding into complex tertiary structures. Their high specificity and low toxicity render them a valid alternative to antibodies for in vivo targeted recognition as therapeutics or delivery agents for nanoparticles, small interfering RNAs, chemotherapeutics and molecular imaging probes. Here we report a novel PDGFRß-specific antagonist that is a nuclease resistant RNA-aptamer, named Gint4. The aptamer, generated by cell-SELEX on glioma U87MG cells, specifically binds to the extracellular domain of the human PDGFRß (Kd, 10.4 nM). Following binding, the aptamer inhibits ligand-dependent receptor activation, including Erk and Akt phosphorylation, cell growth, migration and invasion. Further, we demonstrated that stimulation of glioma cells with the PDGFRß specific ligand, PDGF-BB, caused EGFR transactivation that was hampered by treatment with both the Gint4 aptamer as well as with CL4, the RNA aptamer that we have previously generated as an high affinity inhibitor of EGFR. Interestingly, the combined treatment of glioma cells with the anti-PDGFRß Gint.4 aptamer together with the anti-EGFR CL4 aptamer strongly cooperates in inducing inhibition of cell survival and growth. Moreover, we demonstrated that Gint4 rapidly and specifically internalizes within the target cells thus it can be used as cargo for tissue specific internalization. Citation Format: Simona Camorani, Carla Lucia Esposito, Silvia Catuogno, Anna Rienzo, Margherita Iaboni, Gerolama Condorelli, Vittorio de Franciscis, Laura Cerchia. Targeting of PDGFRβ with a novel RNA-aptamer inhibits glioma cell survival, growth, invasion and enhances antitumor activity of an anti-EGFR aptamer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2013-3035

Targeting Treatment-Naïve CML Stem/Progenitor Cells From Imatinib-Nonresponders with JAK2 (BMS-911543)/ABL Inhibitor Combination Therapy

AbstractAbstract 1339Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a BCR-ABL fusion gene with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Deregulated tyrosine kinase activity of BCR-ABL perturbs several intracellular signaling pathways, including the JAK2/STAT5 pathway. In particular, JAK2 physically interacts with the C-terminal region of BCR-ABL and is one of the most prominent targets of BCR-ABL. Recent introduction of imatinib mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on treatment of chronic phase CML. However, early relapse, acquired drug resistance and ineffectiveness in eliminating leukemic stem cells remain impediments to successful treatment for many patients. Improved treatment approaches to prevent development of resistant subclones, by combined targeting of other key molecular elements active in CML stem cells, are thus clearly needed. One candidate is a complex between the oncoproteins encoded by Abelson helper integration site-1 (AHI-1), BCR-ABL and JAK2 that we recently identified. This complex contributes to the transforming activity of BCR-ABL and also plays a key role in the IM response/resistance of primary CML stem/progenitor cells. We have therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might be more effective in eliminating treatment-naïve CML stem/progenitor cells in patients destined to develop resistance to TKI monotherapy. Several JAK2 inhibitors are currently in various stages of clinical trials, but their nonspecific, off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of a new, highly specific JAK2 inhibitor (BMS-911543) in combination with a number of TKIs, including IM, dasatinib and nilotinib. First, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. Also, in colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents, especially in medium-sized colonies (50–500 cells per colony, 2–3 fold, p<0.05). To determine whether the results obtained following the simultaneous targeting of BCR-ABL and JAK2 in CML cell lines would extend to primary primitive CML cells and whether this combined BCR-ABL-JAK2 targeting approach could also be therapeutically effective for CML patients who do not respond adequately to treatment with a single TKI, we investigated primitive CML cells obtained at diagnosis from seven CML patients who were classified retrospectively as IM-nonresponders, after initiation of IM therapy. Similar to our cell line studies, intracellular staining analysis showed that combined exposure of CD34+CML cells (n=3) to BMS-911543 and a TKI produced a deeper and more prolonged suppression of pSTAT5 activity than a single agent (40–46% suppression for the combinations vs. 15–20% suppression for the single agents at 72 hrs, p<0.05). Combination treatments also caused greater inhibition in colony growth of these cells compared to single treatments (74–86% vs. 40–50%, p<0.05). Interestingly, the combination of BMS-911543 and a TKI almost completely inhibits BFU-E colony formation as compared to treatment with TKI alone (92–100% vs.63–66%, p<0.01). CFU-GM colonies were also more significantly reduced as a result of combination treatment, compared to single agents (49–71% vs. 30–39%, p<0.01). Furthermore, long-term culture-initiating cell (LTC-IC) assays showed that the more primitive cells were also more significantly eliminated by combination treatments than by single agents (2–5 fold, p<0.05), indicating the merit of combination therapy in targeting very primitive cells. Most importantly, our CFC data indicate that BMS-911543 has far less toxicity on normal bone marrow CD34+ cells (n=4) than CML samples (2–3 fold, p<0.05). This study suggests a rational strategy for improving therapeutic outcomes in CML IM-nonresponders by simultaneously targeting both BCR-ABL and JAK2 activities in primary CML stem/progenitor cells. Disclosures:Lorenzi:Bristol-Myers Squibb: Employment, Equity Ownership. Woolfson:Bristol-Myers Squibb: Employment, Equity Ownership.

The evolving field of tyrosine kinase inhibitors in the treatment of thyroid carcinoma

The outstanding progress in the molecular basis of thyroid carcinoma has offered a tool for the development of new drugs,mainly tyrosine kinase inhibitors (TKIs).A number of TKIs are now being applied to the treatment of thyroid carcinomas.Recent clinical trials of TKIs in patients with advanced thyroid cancer have shown some promising preliminary results.This review summarizes the most relevant achievements in the field and the challenges we are facing. Key words: Thyroid carcinoma; Targeted therapy; Tyrosine kinase inhibitors

Bilan des inhibiteurs de protéine tyrosine kinase dans le traitement des cancers

The tyrosine kinase inhibitors (TKI) are small molecules of low molecular weight that inhibit tyrosine kinases, enzymes responsible for the activation of signal transduction cascades. Currently, a number of TKI received approval in various cancers, while others are in clinical development process: TKI are specifically clinically active when they target a tyrosine kinase (TK) with constitutional activity subsequent to a mutation, being then a master-gene driving transformation and tumour progression. Already, this drug-family provides a major therapeutic weapon against cancer.

Reversing multidrug resistance by tyrosine kinase inhibitors

Recently, a large number of tyrosine kinase inhibitors (TKIs) have been developed as anticancer agents. These TKIs can specifically and selectively inhibit tumor cell growth and metastasis by targeting various tyrosine kinases and thereby interfering with cellular signaling pathways. The therapeutic potential of TKIs has been hindered by multidrug resistance (MDR), which is commonly caused by overexpression of ATP-binding cassette (ABC) membrane transporters. Interestingly, some TKIs have also been found to reverse MDR by directly inhibiting the function of ABC transporters and enhancing the efficacy of conventional chemotherapeutic drugs. In this review, we discuss ABC transporter-mediated MDR to TKIs and MDR reversal by TKIs.

Experience on surgical treatment of gastrointestinal stromal tumor of the stomach

In spite of their rarity, gastrointestinal stromal tumors (GISTs) represent a complex clinical problem, mainly diagnostic and therapeutic, for their unpredictable biological course and their long-term prognosis, the most involved site being the stomach. Although a great number of tyrosine-kinase inhibitors has been developed for blocking their proliferative pathways (constitutive CD117 and PDGFRa activation), surgical treatment still remains the only curative one. Nevertheless, their particular non-lymphatic spread and their tendency to peritoneal seeding have emphasized technical issues that are still greatly debated. The definition of the best surgical procedure aiming at the complete R0 resection of the tumor has changed in the recent years and, with the improvement of laparoscopic techniques, the minimally invasive approach of gastric GIST has become feasible in most cases. In this paper we present our experience on surgical treatment of 43 gastric GISTs observed from 2001 to 2008 taken from our case study (75 patients from 1994). The risk class, treatment and long-term follow-up (mean 36 months) has been analyzed. All patients underwent a surgical procedure; 10 of them were also treated with molecular tyrosine-kinase inhibitors as adjuvant treatment. Overall survival at 60 months was 89.3%, with a disease-free survival of 87.68%.

Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance

In recent years, a number of TKIs (tyrosine kinase inhibitors) targeting epidermal growth factor receptor (EGFR) family have been synthesized and some have been approved for clinical treatment of cancer by the FDA. We recently reported a new pharmacological action of the 4-anilinoquinazoline derived EGFR TKIs, such as lapatinib (Tykerb®) and erlotinib (Tarceva®), which significantly affect the drug resistance patterns in cells expressing the multidrug resistance (MDR) phenotype. Previously, we showed that lapatinib and erlotinib could inhibit the drug efflux function of P-glycoprotein (P-gp, ABCB1) and ABCG2 transporters. In this study, we determined if these TKIs have the potential to reverse MDR due to the presence of the multidrug resistance protein 7 (MRP7, ABCC10). Our results showed that lapatinib and erlotinib dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to several established MRP7 substrates, specifically docetaxel, paclitaxel, vinblastine and vinorelbine, whereas there was no or a less effect on the control vector transfected HEK293 cells. [3H]-paclitaxel accumulation and efflux studies demonstrated that lapatinib and erlotinib increased the intracellular accumulation of [3H]-paclitaxel and inhibited the efflux of [3H]-paclitaxel from MRP7-transfected cells but not in the control cell line. Lapatinib is a more potent inhibitor of MRP7 than erlotinib. In addition, the Western blot analysis revealed that both lapatinib and erlotinib did not significantly affect MRP7 expression. We conclude that the EGFR TKIs, lapatinib and erlotinib reverse MRP7-mediated MDR through inhibition of the drug efflux function, suggesting that an EGFR TKI based combinational therapy may be applicable for chemotherapeutic practice clinically.