Aberrant Tyrosine Kinase Activity Research Articles

New Inhibitors of Bcr-Abl Based on 2,6,9-Trisubstituted Purine Scaffold Elicit Cytotoxicity in Chronic Myeloid Leukemia-Derived Cell Lines Sensitive and Resistant to TKIs.

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 μM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 μM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 μM) compared to 11b-f (GI50 = 6.4-11.5 μM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

Abstract 3003: Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling

Abstract Background: Glioblastoma (GBM) is one of the most lethal forms of cancer. The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which upon activation, are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling resulting in hyperactive RTK signaling. We demonstrated that the alternative splicing of the tumor suppressor Annexin A7 (ANXA7) supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. Exon six of ANXA7 can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). Methods: We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates first in U251 malignant glioma followed by stem-like brain tumor initiating cells (BTICs) originating from patient-derived xenografts (PDXs). U251s and primary BTICs (JX14) were transduced with lentivirus to overexpress ANXA7 and GFP for enrichment by flow cytometry. JX14 empty vector (EV; I2 only) and I1 (I2 and I1 expressing) cells were plated onto Geltrex and ligand starved overnight in serum-free media to maintain BTICs. We assessed EGFR protein levels following EGF stimulation in a time dependent manner. Immunofluorescence experiments were conducted to measure EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway, which included the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Results: Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (Rab11: R2=0.9267, P<0.0001). Immunoblotting revealed a significant reduction in EGFR protein in I1 cells only. Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). ANXA7 siRNA in EV cells did not alter EGFR trafficking, which implicates I2 as nonfunctional. On the other hand, ANXA7 KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1. Loss of I1 also shifted the degradation of EGFR back towards recycling. Conclusion: GBMs express I2, which supports EGFR hyperactivity by favoring receptor recycling. I1 overexpression significantly reduced EGFR signaling by promoting degradation. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for BTICs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition. Citation Format: Aran Merati, Sindhu Nair, Rajani Rajbhandari, Nicholas Johnson, Markus Bredel. Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3003.

CD45 inhibition in myeloid leukaemia cells sensitizes cellular responsiveness to chemotherapy

Myeloid malignancies are a group of blood disorders characterized by the proliferation of one or more haematopoietic myeloid cell lineages, predominantly in the bone marrow, and are often caused by aberrant protein tyrosine kinase activity. The protein tyrosine phosphatase CD45 is a trans-membrane molecule expressed on all haemopoietic blood cells except that of platelets and red cells. CD45 regulates various cellular physiological processes including proliferation, apoptosis, and lymphocyte activation. However, its role in chemotherapy response is still unknown; therefore, the aim of this study was to investigate the role of CD45 in myeloid malignancies in terms of cellular growth, apoptosis, and response to chemotherapy. The expression of CD45 on myeloid leukaemia primary cells and cell lines was heterogeneous with HEL and OCI-AML3 cells showing the highest level. Inhibition of CD45 resulted in increased cellular sensitivity to cytarabine and ruxolitinib, the two main therapies for AML and MPN. Bioinformatics analysis identified genes whose expression was correlated with CD45 expression such as JAK2, ACTR2, THAP3 Serglycin, and PBX-1 genes, as well as licensed drugs (alendronate, allopurinol, and balsalazide), which could be repurposed as CD45 inhibitors which effectively increases sensitivity to cytarabine and ruxolitinib at low doses. Therefore, CD45 inhibition could be explored as a potential therapeutic partner for treatment of myeloid malignancies in combination with chemotherapy such as cytarabine especially for elderly patients and those showing chemotherapy resistance.

Treatment failure with first line imatinib for patients with chronic myeloid leukaemia in Kenya vs high-income countries.

e19068 Background: Chronic Myeloid Leukemia (CML) occurs in all age groups but predominantly in adults. The disease pathogenesis is typified by the presence of BCR-ABL fusion protein coded for by BCR-ABL gene, a product of reciprocal translocation between the long arms of chromosomes 9 and 22 denoted: t(9;22)(q34;q11). This results in leukaemogenic aberrant tyrosine kinase activity. The clinical course is characterized by three phases namely – chronic stable phase, accelerated phase and acute (blastic phase). Imatinib mesylate, an inhibitor of BCR-ABL was approved for treatment of CML and revolutionized its treatment and outcome. There is however a failure rate on imatinib of about 15 % that is partially addressed by second- and third-line tyrosine kinase inhibitors. Methods: We retrospectively collected data on patients treated at the Glivec International Patient Assistance Program (GIPAP), later Max Access Solutions (MAS) clinics at the Nairobi Hospital. Patient demographics, date of diagnosis, disease phase, and date of commencement of imatinib were documented. Treatment failure/switch, and reason were also documented. Results: A total of 1347 patients were included. Mean age was 44.12 years, males were 748 (55.5%), females 599 ((44.5%). All except two (1345) were initiated on imatinib, 206 (15.3%) were switched to second-line therapy due to various reasons including 31(2.3%) due to adverse events, 148 (11%) treatment failure, 15 (1.1%) due to unspecified reasons and 1 due to drug stock-outs. Second line medications were given to 204 patients: 100 (49%) dasatinib, 50 (24.5%) bosutinib, 38 (18.6%) nilotinib, 16 (7.8%) ponatinib. Deaths or losses to follow-up were 181 (13.4%). Conclusions: Overall about 15% failed on imatinib first-line therapy, similar to the experience from high-income countries. The most preferred second-line TKI was dasatinib, based on availability and toxicity profile.

Novel pharmacological and dietary approaches to target mTOR in B-cell acute lymphoblastic leukemia.

High-risk subtypes of B-cell acute lymphoblastic leukemia (B-ALL) are frequently associated with aberrant activation of tyrosine kinases (TKs). These include Ph+ B-ALL driven by BCR-ABL, and Ph-like B-ALL that carries other chromosomal rearrangements and/or gene mutations that activate TK signaling. Currently, the tyrosine kinase inhibitor (TKI) dasatinib is added to chemotherapy as standard of care in Ph+ B-ALL, and TKIs are being tested in clinical trials for Ph-like B-ALL. However, growth factors and nutrients in the leukemia microenvironment can support cell cycle and survival even in cells treated with TKIs targeting the driving oncogene. These stimuli converge on the kinase mTOR, whose elevated activity is associated with poor prognosis. In preclinical models of Ph+ and Ph-like B-ALL, mTOR inhibitors strongly enhance the anti-leukemic efficacy of TKIs. Despite this strong conceptual basis for targeting mTOR in B-ALL, the first two generations of mTOR inhibitors tested clinically (rapalogs and mTOR kinase inhibitors) have not demonstrated a clear therapeutic window. The aim of this review is to introduce new therapeutic strategies to the management of Ph-like B-ALL. We discuss novel approaches to targeting mTOR in B-ALL with potential to overcome the limitations of previous mTOR inhibitor classes. One approach is to apply third-generation bi-steric inhibitors that are selective for mTOR complex-1 (mTORC1) and show preclinical efficacy with intermittent dosing. A distinct, non-pharmacological approach is to use nutrient restriction to target signaling and metabolic dependencies in malignant B-ALL cells. These two new approaches could potentiate TKI efficacy in Ph-like leukemia and improve survival.

Cholesterol suppresses GOLM1-dependent selective autophagy of RTKs in hepatocellular carcinoma.

Aberrant activation of receptor tyrosine kinases (RTKs) and the subsequent metabolic reprogramming play critical roles in cancer progression. Our previous study has shown that Golgi membrane protein 1 (GOLM1) promotes hepatocellular carcinoma (HCC) metastasis by enhancing the recycling of RTKs. However, how this RTK recycling process is regulated and coupled with RTK degradation remains poorly defined. Here, we demonstrate that cholesterol suppresses the autophagic degradation of RTKs in a GOLM1-dependent manner. Further mechanistic studies reveal that GOLM1 mediates the selective autophagy of RTKs by interacting with LC3 through an LC3-interacting region (LIR), which is regulated by a cholesterol-mTORC1 axis. Lowering cholesterol by statins improves the efficacy of multiple tyrosine kinase inhibitors (TKIs) invivo. Our findings indicate that cholesterol serves as a signal to switch GOLM1-RTK degradation to GOLM1-RTK recycling and suggest that lowering cholesterol by statin may be a promising combination strategy to improve the TKI efficiency in HCC.

Current Management of Chronic Myeloid Leukemia Myeloid Blast Phase.

Despite the major advancements in the management of chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) remains a major therapeutic challenge. BC can be myeloid, lymphoid, or mixed lineage with myeloid BC being the most common type. BC in CML is mediated by aberrant tyrosine kinase activity of the BCR::ABL fusion protein. The introduction of BCR::ABL tyrosine kinase inhibitor (TKI) has been a gamechanger in the treatment of CML and there has been a significant reduction in the incidence of BC. The main treatment goal in BC is to achieve a second CP and consolidate that with an allogeneic stem cell transplantation (SCT) in eligible patients. The outcomes in BC remain dismal even in the current era. In this review, we provide an overview of the biology and current therapeutic approach in myeloid BC.

Focal adhesion kinase inhibition synergizes with nab-paclitaxel to target pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a very lethal disease, with minimal therapeutic options. Aberrant tyrosine kinase activity influences tumor growth and is regulated by phosphorylation. We investigated phosphorylated kinases as target in PDAC.MethodsMass spectrometry-based phosphotyrosine proteomic analysis on PDAC cell lines was used to evaluate active kinases. Pathway analysis and inferred kinase activity analysis was performed to identify novel targets. Subsequently, we investigated targeting of focal adhesion kinase (FAK) in vitro with drug perturbations in combination with chemotherapeutics used against PDAC. Tyrosine phosphoproteomics upon treatment was performed to evaluate signaling. An orthotopic model of PDAC was used to evaluate the combination of defactinib with nab-paclitaxel.ResultsPDAC cell lines portrayed high activity of multiple receptor tyrosine kinases to various degree. The non-receptor kinase, FAK, was identified in all cell lines by our phosphotyrosine proteomic screen and pathway analysis. Targeting of this kinase with defactinib validated reduced phosphorylation profiles. Additionally, FAK inhibition had anti-proliferative and anti-migratory effects. Combination with (nab-)paclitaxel had a synergistic effect on cell proliferation in vitro and reduced tumor growth in vivo.ConclusionsOur study shows high phosphorylation of several oncogenic receptor tyrosine kinases in PDAC cells and validated FAK inhibition as potential synergistic target with Nab-paclitaxel against this devastating disease.

Abstract 4682: Influence of PI3K/AKT pathway on imatinib mesylate treatment outcome in chronic myeloid leukemia patients

Abstract Chronic myeloid leukemia (CML) is a common myeloproliferative disorder characterized by cytogenetic abnormality [t(9;22)(q34;q11)] that generates BCR-ABL oncogene with aberrant tyrosine kinase activity, the driver event in CML origin. Tyrosine kinase inhibitors (TKIs) are remarkably effective; yet, treatment failure is common in some cases due to the accumulation of mutations in BCR-ABL itself and other genes causing drug resistance/relapse, which indicates that BCR-ABL inhibition alone may not be sufficient to combat CML. The PI3K/Akt/mTOR signaling pathway downstream of BCR-ABL has been proven to be an important survival mechanism in leukemias. In vitro studies demonstrated that targeting this pathway could be a viable strategy, but needs further validation in CML patients. In this study, we examined the critical PI3K/AKT/mTOR pathway genes (PIK3CA, PTEN, AKT1, SHIP1, mTOR, and FOXO3A) in CML patients (n=51) using qPCR method at diagnosis and after treatment, and analyzed with respect to BCR-ABL status, and JAK2, STAT5, and STAT3 levels. Interestingly, significant upregulation of AKT1 (p=0.012) and PIK3CA (p=0.026) and downregulation of PTEN (P=0.06) and SHIP1 (p=0.039) was observed in samples at diagnosis compared to their post imatinib treated. Remarkably, these elevated AKT1 and PI3KCA transcript levels were significantly associated with increased BCR-ABL and JAK2 transcript levels, but not STAT3 and STAT5 suggesting PI3K pathway as a prominent target in CML. However, with respect to the clinical phase and imatinib response, none of the genes showed significant differences except for PTEN, which was significantly downregulated in advanced phase (p<0.042) and in cases with poor imatinib response. Overall, these results indicate a critical role for PI3K pathway in CML and its maintenance. Citation Format: Swathi Banapuram, Manjula Gorre, Raghunadha Rao Digumarti, Vishnupriya Satti, Komaraiah Palle, Sandhya Annamaneni. Influence of PI3K/AKT pathway on imatinib mesylate treatment outcome in chronic myeloid leukemia patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4682.

Abstract 4219: Efficacy of CR13626, a novel oral brain penetrant multi-kinase inhibitor, in a mouse model of glioblastoma

Abstract BACKGROUND: Currently there is no cure for glioblastoma (GBM), the most malignant primary brain cancer. Glioblastoma etiopathogenesis involves mutations and alterations of key signaling pathways including tyrosine kinases (TKs). Notably, aberrant activation of receptor tyrosine kinases (RTKs) such as EGFR (amplified in 40% of glioblastoma cases) and KDR represents one of the driving forces for cellular proliferation and angiogenesis. The ability to cross the blood brain barrier (BBB) and appropriate pharmacodynamics (target selectivity) and pharmacokinetics (brain penetration) are critical issues for the generation of potential drug candidates against glioblastoma. CR13626 is a novel brain penetrant small molecule able to inhibit different TKs relevant to the development of this cancer. The present study describes the in vitro and in vivo properties of CR13626 and its antitumor activity in a mouse model of glioblastoma. METHODS: We identified the TKs targeted by CR13626 against a panel of 173 kinase enzymes. The effects of CR13626 on cellular proliferation were assessed in different 2D human GBM cell lines (U87MG, U373, U87MG vIII) by ViaCount, and in U87MG 3D spheroids by CellTiter-Glo 3D viability assay. The antitumor activity in vivo was determined in an orthotopic mouse model of GBM based on the injection of U87MG-Luciferase cells in nude mice. Animals were orally treated with CR13626 (50 mg/kg/daily) for 10 days, starting on day 9 post-implantation. Measurement of bioluminescence (BLI) at day 19 (end of dosing) and during follow-up (days 26-33) allowed the monitoring of tumor progression. CR13626 pharmacokinetics and brain exposure were assessed by LC/MS/MS in plasma and brain homogenate tissues of CD1 and tumor-bearing nude mice. RESULTS: CR13626 potently inhibited tyrosine kinases relevant to GBM development, with IC50 values in the nanomolar range: FYN (69 nM), YES (3.6 nM), KDR (82 nM) and EGFR (6 nM). CR13626 reduced the growth of different human glioblastoma cell lines with GI50 values in the range 1-3 µM. In vivo, CR13626 treatment led to a time-dependent reduction of tumor growth, reaching 60% on the last BLI evaluation 33 days post-implantation (i.e. 15 days after the end of dosing). A 25% significant increase in the median survival of animals compared to the vehicle group was also observed. The antitumor effects of CR13626 were in line with the exposure of tumor-bearing mice to the compound. Pharmacokinetic studies in CD1 mice showed good oral bioavailability (72%) and brain penetration (brain/plasma ratio of 1.4) for CR13626. CONCLUSION: The ability of CR13626 to cross the BBB without being a substrate of efflux transporters that mediate tumor resistance, to simultaneously inhibit the activity of different TKs involved in GBM development, and to reduce tumor growth eventually leading to an increased survival of animals, warrant its further development as a drug candidate in glioblastoma. Citation Format: Chiara Galimberti, Tiziana Piepoli, Giuseppe M. Montagna, Silvia Zerbi, Ornella Letari, Roberto Artusi, Milena Colovic, Stefano Persiani, Gianfranco Caselli, Lucio C. Rovati. Efficacy of CR13626, a novel oral brain penetrant multi-kinase inhibitor, in a mouse model of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4219.

SRC Kinase in Glioblastoma News from an Old Acquaintance.

Glioblastoma multiforme (GBM) is one of the most recalcitrant brain tumors characterized by a tumor microenvironment (TME) that strongly supports GBM growth, aggressiveness, invasiveness, and resistance to therapy. Importantly, a common feature of GBM is the aberrant activation of receptor tyrosine kinases (RTKs) and of their downstream signaling cascade, including the non-receptor tyrosine kinase SRC. SRC is a central downstream intermediate of many RTKs, which triggers the phosphorylation of many substrates, therefore, promoting the regulation of a wide range of different pathways involved in cell survival, adhesion, proliferation, motility, and angiogenesis. In addition to the aforementioned pathways, SRC constitutive activity promotes and sustains inflammation and metabolic reprogramming concurring with TME development, therefore, actively sustaining tumor growth. Here, we aim to provide an updated picture of the molecular pathways that link SRC to these events in GBM. In addition, SRC targeting strategies are discussed in order to highlight strengths and weaknesses of SRC inhibitors in GBM management, focusing our attention on their potentialities in combination with conventional therapeutic approaches (i.e., temozolomide) to ameliorate therapy effectiveness.

Immunomodulatory Activity of Tyrosine Kinase Inhibitors to Elicit Cytotoxicity Against Cancer and Viral Infection.

Tyrosine kinase inhibitors (TKIs) of aberrant tyrosine kinase (TK) activity have been widely used to treat chronic myeloid leukemia (CML) for decades in clinic. An area of growing interest is the reported ability of TKIs to induce immunomodulatory effects with anti-tumor and anti-viral activity, which appears to be mediated by directly or indirectly acting on immune cells. In selected cases of patients with CML, TKI treatment may be interrupted and a non-drug remission may be observed. In these patients, an immune mechanism of increased anti-tumor cytotoxic activity induced by chronic administration of TKIs has been suggested. TKIs increase some populations of natural killer (NK), NK-LGL, and T-LGLs cells especially in dasatinib treated CML patients infected with cytomegalovirus (CMV). In addition, dasatinib increases responses against CMV and is able to inhibit HIV replication in vitro. Recent studies suggest that subclinical reactivation of CMV could drive expansion of specific subsets of NK- and T-cells with both anti-tumoral and anti-viral function. Therefore, the underlying mechanisms implicated in the expansion of this increased anti-tumor and anti-viral cytotoxic activity induced by TKIs could be a new therapeutic approach to take into account against cancer and viral infections such as HIV-1 infection. The present review will briefly summarize the immunomodulatory effects of TKIs on T cells, NKs, and B cells. Therapeutic implications for modulating immunity against cancer and viral infections and critical open questions are also discussed.

Reduced Expression of Sprouty1 Contributes to the Aberrant Proliferation and Impaired Apoptosis of Acute Myeloid Leukemia Cells.

In most of the acute myeloid leukemia patients there is an aberrant tyrosine kinase activity. The prototype of Sprouty proteins was originally identified in Drosophila melanogaster as antagonists of Breathless, the mammalian ortholog of fibroblast growth factor receptor. Usually, SPRY family members are inhibitors of RAS signaling induced by tyrosine kinases receptors and they are implicated in negative feedback processes regulating several intracellular pathways. The present study aims to investigate the role of a member of the Sprouty family, Sprouty1, as a regulator of cell proliferation and growth in patients affected by acute myeloid leukemia. Sprouty1 mRNA and protein were both significantly down-regulated in acute myeloid leukemia cells compared to the normal counterpart, but they were restored when remission is achieved after chemotherapy. Ectopic expression of Sprouty1 revealed that it plays a key role in the proliferation and apoptotic defect that represent a landmark of the leukemic cells. Our study identified Sprouty1 as negative regulator involved in the aberrant signals of adult acute myeloid leukemia. Furthermore, we found a correlation between Sprouty1 and FoxO3a delocalization in acute myeloid leukemia (AML) patients at diagnosis, suggesting a multistep regulation of RAS signaling in human cancers.

Abstract 4887: Recurrent NRG1 rearrangements in Caucasian pulmonary mucinous adenocarcinoma: results from an Italian multi-center cohort

Abstract Invasive Mucinous Adenocarcinoma (IMA) is a rare histotype of lung adenocarcinoma associated with an unfavorable prognosis due to the lack of effective treatment. The NRG1 rearrangement is a new subtype-specific molecular feature of IMA and acts as a strong oncogenic inductor of the aberrant tyrosine kinase activity of ErbB2/ErbB3 heterodimers through PI3K-AKT/MAPK cellular cascades. We recently described for the first time the occurrence of NRG1 rearrangements in 31% of Caucasian lung IMAs and highlighted a strong association between NRG1 rearrangements and ErbB3 activation. Here we extended our lung IMA samples cohort by enrolling a total of 71 patients from three different Italian Centers and collected clinical-pathological information and molecular profile, included the mutational status of KRAS, EGFR and ALK genes. We screened all samples by fluorescent in situ hybridization (FISH) for the detection of putative NRG1 rearrangements and by immunohistochemistry (IHC) for the expression of phosphorylated-ErbB3 (pErbB3) receptor. Finally, we customized a new targeted RNA Custom Panel to detect all 9 NRG1-fusion variants published to date to molecular characterize the NRG1 fusion variants in NRG1 rearranged IMAs. Results showed NRG1 rearrangements in 32% of lung IMAs, displaying both NRG1 FISH split signals and deletions of the 5' portion of the gene. IHC confirmed our previous findings of association between pErbB3 immunoreactivity and NRG1 rearrangements, and the heterogeneity of fluorescent signal distribution and immunostaining along the tissue sections. The CD74-NRG1 remains the most common fusion variant identified in lung IMA samples. Correlation analysis among clinical-pathological data, pErbB3 expression and NRG1 rearrangements are ongoing. Our results confirm the usefulness of IHC/FISH combined approach for NRG1 broken tumors identification and highlight the role of NRG1 rearrangement as master molecular marker of lung IMAs, potentially useful to select patients for the emerging target therapies. Citation Format: Domenico Trombetta, Paolo Graziano, Angelo Sparaneo, Giulio Rossi, Antonio Rossi, Marcello Tiseo, Massimo Di Maio, Federico P. Fabrizio, Maria C. Manzorra, Flavia Centra, Leonarda Di Candia, Marco Audisio, Evaristo Maiello, Vito M. Fazio, Lucia A. Muscarella. Recurrent NRG1 rearrangements in Caucasian pulmonary mucinous adenocarcinoma: results from an Italian multi-center cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4887.

Downregulation of Mir-142 Promotes Leukemia Growth in Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL): A Possible Novel Therapeutic Target?

The Philadelphia (Ph) chromosome or t(9;22) results in the generation of a fusion gene, namely BCR/ABL1, which encodes a chimeric protein with aberrant tyrosine kinase activity that drives leukemia cell growth and survival. This molecular/cytogenetic aberration occurs in ~20%-30% of ALL cases and confers poor prognosis. Ph+ ALL patients (pts) are often referred for allogeneic hematopoietic stem cell transplantation (alloHCT), although more recently BCR-ABL-specific tyrosine-kinase inhibitors (TKIs) and immunotherapeutic approaches seemingly induced long-term remission in some patients. Nevertheless, it is still a challenge to determine which Ph+ ALL of the pts could be treated more conservatively without alloHCT. Thus identification of new prognostic biomarkers and/or therapeutic targets may be helpful. Regulation of short non-coding microRNAs(miRNAs) associated with initiation and progression of acute leukemia has been reported. miR-142(both miR-142-3p and miR-142-5p) is expressed at a relatively high level in hematopoietic tissue, and plays a role in myeloid lineage differentiation. In fact, low miR-142-3p expression was associated with myeloid differentiation failure, and miR-142 mutations was reported to promote acute myeloid leukemia (AML). More recently, Kramer et al demonstrated a role of miR-142 in lymphopoiesis by showing that miR-142 deficiency impaired B cell production in a miR-142 knock-out(ko) mouse model (Blood. 2015).Here, we first investigated if miR-142 levels were altered in ALL pts. Analysis of a publically available miRNA expression dataset(GSE23024) showed lower level of miR-142-3p, but not miR-142-5p in Ph+ ALL pts(n=10) vs. healthy donors(n=7;p=0.0093); while no significant differences were observed in Ph- pre-B ALL pts(n=61) vs. healthy donors (n=7). In ALL Tg(P190-BCR/ABL) transgenic mice(Ph+ ALL; Nature. 1990), we found bone marrow (BM) miR-142-3p level to be ~2.3-fold lower than those in the wild-type (wt) controls(p=0.036). Compared to wt mice, Ph+ ALL mice showed significantly lower miR-142-3p level in all the immunophenotypically identified BM lymphoid subpopulations, including progenitor B (pro-B, B220+CD19+CD43+IgM-,~19.1-fold lower,p<0.0001), precursor B (pre-B, B220+CD19+CD43-IgM-,~9.7-fold lower, p<0.0001), and other immature B (B220lowCD19+CD43-IgM+, ~2.4-fold lower, p<0.001) cells, except for mature B (B220highCD19+CD43-IgM+) cells. Ph+ ALL mice exhibited a miR-142-3p gradient expression pattern following the lymphoid differentiation hierarchy, with the lowest levels found in the pro-B and pre-B populations. These results prompted us to hypothesize that, loss of miR-142 may contribute to primitive B cell expansion possibly due to B cell differentiation blockage in Ph+ ALL mice. To prove this, we crossed miR-142 double knock-out (d-ko)mice with Ph+ ALL mice to generate miR-142(ko)Tg(P190-BCR/ABL) mice. Homozygous miR-142(d-ko)Tg(P190-BCR/ABL) mice were not viable due to an overly aggressive leukemia phenotype. Heterozygous miR-142(wt/ko)Tg(P190-BCR/ABL) mice had evidence of more rapid expansion of pro-B cells in blood(PB; 47.9% vs. 9.8%, p<0.0001), BM (48.2% vs. 13.2%, p<0.01)and spleen(32.3%vs. 4.4%, p<0.01) at 6 weeks old and a significantly reduced survival(median survival 44 vs.80 days, p<0.0001), compared to miR-142(wt/wt)Tg(P190-BCR/ABL) controls. BM cells (CD45.2) from miR-142(wt/ko)Tg(P190-BCR/ABL) mice (n=4) or miR-142(wt/wt)Tg(P190-BCR/ABL) mice (n=5) were then transplanted into congenic CD45.1 recipient mice (n=18 and n=15 respectively).Recipients of BM cells from miR-142(wt/ko)Tg(P190-BCR/ABL) donors showed increased engraftment (94% vs. 77% in PB at 4 weeks, p<0.0001) and significantly reduced survival(median survival 25 vs. 49 days, p<0.0001), as compared with recipients of BM cells from miR-142(wt/wt)P190-BCR-ABL mice. Finally, upon ex vitro exposure to the TKI nilotinib (5uM for 48 hours), miR-142(wt/ko)Tg(P190-BCR/ABL) BM cells showed reduced apoptosis (7.0% vs.37.5% vs p<0.05) and increased cell viability (66% vs.16.2%, p<0.05) compared with miR-142 (wt/wt)Tg(P190-BCR/ABL) BM cells. In vivo treatment studies with TKI treatment are ongoing and data will be presented at the meeting. In conclusion, miR-142 downregulation promotes rapid Ph+ ALL growth likely by contributing to a blockage of B cell differentiation, and may mediate resistance to TKIs. DisclosuresStein:Celgene: Speakers Bureau; Amgen Inc.: Speakers Bureau. Jin:The National Natural Science Foundation of China: Research Funding; College of Medicine, Zhejiang University: Employment.

Receptor Tyrosine Kinase-Targeted Cancer Therapy.

In the past two decades, several molecular targeted inhibitors have been developed and evaluated clinically to improve the survival of patients with cancer. Molecular targeted inhibitors inhibit the activities of pathogenic tyrosine kinases. Particularly, aberrant receptor tyrosine kinase (RTK) activation is a potential therapeutic target. An increased understanding of genetics, cellular biology and structural biology has led to the development of numerous important therapeutics. Pathogenic RTK mutations, deletions, translocations and amplification/over-expressions have been identified and are currently being examined for their roles in cancers. Therapies targeting RTKs are categorized as small-molecule inhibitors and monoclonal antibodies. Studies are underway to explore abnormalities in 20 types of RTK subfamilies in patients with cancer or other diseases. In this review, we describe representative RTKs important for developing cancer therapeutics and predicting or evaluated resistance mechanisms.

BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia.

The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the −11.7 kb and −0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34− cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.

Abstract 1084: TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis

Abstract Receptor tyrosine kinases (RTKs) are important drivers of cancers. In addition to genomic alterations, aberrant activation of wild type RTKs play an important role in driving cancer progression. However, the underlying mechanisms of how RTKs drive prostate cancer remains incompletely characterized. Here we show that non-proteolytic ubiquitination of RTK regulates its kinase activity and contributes to RTK-mediated prostate cancer metastasis. TRAF4, a RING domain E3 ubiquitin ligase, is highly expressed in metastatic prostate cancer. We demonstrated here that it is a key player in regulating RTK mediated prostate cancer metastasis. We further identified TrkA, a neurotrophin receptor tyrosine kinase, as a TRAF4-targeted ubiquitination substrate that promotes cancer cell invasion and genetic ablation of TrkA abolished TRAF4-dependent cell invasion. TRAF4 promoted atypical K27 and K29-linked ubiquitination at the TrkA kinase domain and increased its kinase activity. Mutation of TRAF4-targeted ubiquitination sites abolished TrkA tyrosine auto-phosphorylation and its interaction with downstream effector proteins. TRAF4 knockdown also suppressed nerve growth factor-stimulated TrkA downstream p38 MAPK activation and subsequent invasion-associated gene expression. Furthermore, elevated TRAF4 levels significantly correlated with increased NGF-stimulated invasion-associated gene expression in prostate cancer patients, indicating this signaling axis is significantly activated during oncogenesis. Our results revealed a post-translational modification mechanism contributing to aberrant non-mutated receptor tyrosine kinase activation in cancer cells. Citation Format: Ramesh Singh, Dileep Karri, Jiangyong Shao, Subhamoy Dasgupta, Shixia Huang, Dean P. Edwards, Bert W. O'Malley, Ping Yi. TRAF4-mediated NGF receptor TrkA ubiquitination regulates prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1084.

Cisplatin-induced non-canonical endocytosis of EGFR via p38 phosphorylation of the C-terminal region containing Ser-1015 in non-small cell lung cancer cells.

The aberrant activation of receptor tyrosine kinases (RTKs) is associated with tumor initiation in various types of human cancer, including non-small cell lung cancers (NSCLCs). Tyrosine kinase-independent non-canonical RTK regulation has also been investigated in tumor malignant alterations, including cellular stress responses. It was recently reported that the phosphorylation of epidermal growth factor receptor (EGFR) at C-terminal Ser-1015 serves a critical role in growth factor and cytokine signaling. In the present study, the role of non-canonical EGFR regulation has been investigated in NSCLC cells treated with cisplatin, a common chemotherapeutic agent. Cisplatin-induced p38 activation triggered the Ser-1015 phosphorylation of EGFR, with similar kinetics to previously reported Ser-1047 phosphorylation, in a tyrosine kinase-independent manner. In addition, phosphorylation around Ser-1015 triggered endocytosis of a dimer deficient mutant of EGFR. The non-canonical endocytosis of EGFR monomers was primarily controlled by the region around Ser-1015 only; however, Ser-1047 on internalized EGFR was equally phosphorylated. The results of the present study provide mechanistic evidence for the cisplatin-induced non-canonical regulation of EGFR.

A novel multikinase inhibitor R8 exhibits potent inhibition on cancer cells through both apoptosis and autophagic cell death.

Chemotherapy is an important treatment for cancer patients, especially for those with unresectable lesions. Targeted therapy of cancer by specific inhibition of aberrant tyrosine kinase activities in cancer cells with chemically synthesized tyrosine kinase inhibitors (TKIs), shows better responses while less side effects than traditional chemotherapeutic drugs. It is common that cancer cells often exhibit deregulation of several tyrosine kinases simultaneously, multikinase TKIs (MKIs) therefore have greater advantages over single-target TKIs. Currently more MKIs are under developing for better efficacy for different types of cancer. In the present work, we evaluated the in vitro therapeutic potential of a novel MKI, namely R8, with comparison to the clinically available MKI Sunitinib. Results showed that R8 has stronger inhibition on six different types of cancer cell lines with lower IC50 than Sunitinib does. Cell cycle analysis showed that R8 induced significant G0/G1 arrest phase of lung cancer A549 and NCI-H226 cells. The inhibition was also confirmed by colony formation and migration assays in both lung cancer cell lines in a dose-dependent manner. R8 could significantly inhibit the phosphorylation of multiple receptor tyrosine kinases (RTKs) included PDGFRβ, VEGFR2, EGFR and C-Kit, leading to the down-regulation of PI3K-Akt-mTOR signaling. Further analysis revealed that R8 treatment induced more significant apoptosis than Sunitinib did, which might be the consequence of the autophagic cell death. In conclusion, this work suggested R8 to be a promising novel anticancer MKI, and provided the basis for further in vivo investigation on its potential in treatment of lung cancer.