BCR-ABL In Chronic Myelogenous Leukemia Research Articles

Emerging Data on the Safety and Efficacy of Ripretinib for the Treatment of Gastrointestinal Stromal Tumors.

In patients with gastrointestinal stromal tumors (GIST), systemic treatment after disease progression on imatinib is challenging. Sunitinib and regorafenib are approved in the second- and third-line setting, respectively, with activity against certain secondary mutations with comparatively much lower response rates and survival increment compared to imatinib. All three of these drugs were serendipitously found to have activity in GIST, starting with imatinib, which was formulated for its ability to inhibit BCR-ABL in chronic myelogenous leukemia. Ripretinib is a drug that was specifically developed as a more potent KIT tyrosine kinase inhibitor (TKI), with broad-spectrum activity against the mutations encountered in GIST. Encouraging responses in early and later lines of treatment in the Phase 1 trial of ripretinib in GIST led to the rapid development of this novel drug. In a Phase 3 randomized clinical trial with cross-over, ripretinib demonstrated superior PFS and overall survival (OS) in 4th-line treatment and beyond compared to placebo. This established 150 mg once daily ripretinib as the standard of care in this setting. Ripretinib is generally well tolerated, with common adverse effects of hair loss, diarrhea, cramps, fatigue and nausea. The favorable safety profile and efficacy of ripretinib prompted its evaluation in a randomized phase 3 trial in the 2nd-line treatment setting. However, it did not result in a longer PFS duration than sunitinib. Although the efficacy of ripretinib in this unselected patient population was not significantly different from that of sunitinib, the tolerability profile was better. This review article aims to review the efficacy and tolerability profile of ripretinib, together with its role in the setting of unresectable or metastatic GIST.

Identification of A‐B, a Novel Chimeric RNA in Colorectal Cancer

Colorectal cancer (CRC) is the third leading cause of cancer‐related deaths worldwide, affecting more than one hundred thousand men and women in the United States every year. With the incidence of early‐onset (<50 years old) CRC increasing at an alarming rate, there is an urgent and unmet need for early biomarker discovery and the identification of novel therapeutic targets in CRC. Chimeric RNAs and their protein products are well‐established as ideal biomarkers and drug targets for malignant cancers, including BCR‐ABL in chronic myelogenous leukemia and EML4‐ALK in lung cancer. Using RNA‐seq data from The Cancer Genome Atlas (TCGA) database, we have identified a chimeric RNA, denoted as A‐B, present in 45.2% of CRC samples and just 17.6% of adjacent normal margin tissue, but absent in non‐cancer colon tissues. We determined that this chimeric RNA results from cis‐splicing between adjacent genes (cis‐SAGe), and includes a splice variant of the 5’ gene, altering the reading frame of the 3’ gene. This chimeric RNA encodes a novel transmembrane protein, with the alternate reading frame on the extracellular surface, making it an ideal target for antibody therapeutics. After generating an antibody to the chimeric protein and validating its use in immunoblotting, immunoprecipitation, and immunohistochemistry, we found this novel protein is present in patient CRC samples in a distinct, punctate pattern. Overexpression of this chimeric RNA resulted in increased cell proliferation, while siRNA mediated knockdown led to a reduction in cell proliferation. We are currently investigating the function of this chimeric RNA and its biomarker potential in clinical CRC samples.

Dasatinib Promotes the Maturation of Healthy Donors and Chronic Myelogenous Leukemia Patients Derived Dendritic Cells

Dasatinib has been discovered to obtain immunomodulatory effects in addition to the targeting effect towards BCR-ABL in chronic myelogenous leukemia (CML) patients. But the effect of dasatinib on dendritic cells (DCs) is still not fully understood. Objective To investigate the effect of dasatinib on the maturation of monocyte derived dendritic cells (moDCs) from healthy donors (HDs) and CML patients. Method Peripheral blood mononuclear cells (PBMCs) were isolated from HDs (n=14) and CML patients (n=14) who had got remission of MR4.5 with imatinib treatment. The generation of moDCs was completed after 7 days of incubation in DC I medium, and another 3 days of incubation in DC II medium with or without 25nM dasatinib. On the 10th day, the moDCs were harvested and analyzed for the expression of surface markers CD83, CD80, CD86, CD40 and HLA-DR to reflect the maturation of the moDCs by flow cytometry. Results When analyzing the moDCs from all the 14 HDs together, dasatinib significantly enhanced the expression levels of the surface marker CD83 (P=0.039), CD40 (P=0.002) and HLA-DR (P=0.000) on moDCs compared with the control group, but there was no statistically significant difference in CD80 (P=0.073) and CD86 (P=0.897). Differently, when analyzing the moDCs derived from all the 14 patients together, there was no statistically significant difference in the expression levels of CD80 (P=0.086), CD86 (P=0.166), CD83 (P=0.674), CD40 (P=0.574) and HLA-DR (P=0.561) on moDCs between the dasatinib group and the control group. However, moDCs derived from 3 of the 14 patients show the enhanced expression of all the five surface makers with dasatinib administration compared to the control group. Besides, the moDCs derived from 6 of the 14 patients show the enhanced expression of at least one of the five surface markers. Notably, compared with moDCs derived from HD group (n=14), moDCs from patient group (n=14) express lower levels of CD80 (P=0.340), CD86 (P=0.007), CD40 (P=0.010), CD83 (P=0.019) and HLA-DR (P=0.036). Conclusion Dasatinib at the concentration of 25nM can efficiently promote the maturation of HD-derived moDCs in vitro, whereas this effect only occurs in part of the CML patients. And the moDCs from patients generally show much lower levels of the maturation associated surface makers compared with moDCs from HDs. Dasatinib shows potential as a DC adjuvant to be applied in DC-based therapeutic strategies, such as DC vaccine and DC cell-therapy. Disclosures No relevant conflicts of interest to declare.

Deubiquitylase USP25 prevents degradation of BCR-ABL protein and ensures proliferation of Ph-positive leukemia cells.

Fusion genes resulting from chromosomal rearrangements are frequently found in a variety of cancer cells. Some of these are known to be driver oncogenes, such as BCR-ABL in chronic myelogenous leukemia (CML). The products of such fusion genes are abnormal proteins that are ordinarily degraded in cells by a mechanism known as protein quality control. This suggests that the degradation of BCR-ABL protein is suppressed in CML cells to ensure their proliferative activity. Here, we show that ubiquitin-specific protease 25 (USP25) suppresses the degradation of BCR-ABL protein in cells harboring Philadelphia chromosome (Ph). USP25 was found proximal to BCR-ABL protein in cells. Depletion of USP25 using shRNA-mediated gene silencing increased the ubiquitylated BCR-ABL, and reduced the level of BCR-ABL protein. Accordingly, BCR-ABL-mediated signaling and cell proliferation were suppressed in BCR-ABL-positive leukemia cells by the depletion of USP25. We further found that pharmacological inhibition of USP25 induced rapid degradation of BCR-ABL protein in Ph-positive leukemia cells, regardless of their sensitivity to tyrosine kinase inhibitors. These results indicate that USP25 is a novel target for inducing the degradation of oncogenic BCR-ABL protein in Ph-positive leukemia cells. This could be an effective approach to overcome resistance to kinase inhibitors.

Abstract ED01-02: Successes and failures of targeted drug development in pediatric tumors

Abstract Eighty percent of children diagnosed with cancer are cured with multimodality therapy including cytotoxic chemotherapy. However, up to 40% of survivors of childhood cancer have at least one serious chronic medical condition as a consequence of curative therapy. Thus, patients, families, clinicians, researchers, and regulatory agencies enthusiastically endorse the development of targeted therapy to improve survival, decrease life threatening acute toxicity, and late effects of therapy for childhood cancer. Genomic characterization demonstrates that the tumor mutational burden in childhood cancers is low. For children with cancer a small number of drugs, such as NTRK inhibitors have had major unequivocal successes including initial regulatory approvals that include specific dosing in children. Other molecularly targeted drugs have achieved moderate success, however, many have failed despite strong pre-clinical rationale. As of June 2019, 25 drugs have been FDA approved for use in children with cancer including five tyrosine kinase inhibitors (3 BCR-ABL inhibitors and 2 NTRK inhibitors), and one mTOR inhibitor. A single drug, dinutuximab, was approved for a pediatric specific indication in high-risk neuroblastoma. This parallels recent European Medicines Agency authorizations that include indications for treating childhood cancer in 10 of 177 anti-cancer drug approvals. Similar to adults, the era of targeted therapies in children with cancer began with imatinib. Drugs targeting BCR-ABL in chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) in children include imatinib, dasatinib, and nilotinib. Point mutations in the kinase domain of the BCR-ABL fusion protein result in resistance to imatinib. Dasatinib, nilotinib, and ponatinib were developed to overcome resistance. Each has a specific role in caring for children with CML or Ph+ ALL. Ph-like ALL, a high-risk acute leukemia in children and young adults, is characterized by genomic alterations and activation of signaling pathways including JAK/STAT, amenable to inhibition tyrosine kinase inhibitors. The landmark age and histology agnostic global approval of larotrectinib and entrectinib was based on durable objective responses in patients with tumors harboring NTRK fusions enrolled on early phase clinical trials. Larotrectinib has produced dramatic objective responses in children and adults with cancer harboring NTRK fusions including a very rare cancer, infantile fibrosarcoma which is NTRK fusion positive in at least 95% cases. In addition to potent inhibition NTRK fusions, initial clinical trials of larotrectinib included adults and children as well as a pediatric appropriate formulation. Children with brain tumors harboring NTRK1,2,3 or ROS1 fusions have had rapid, durable objective responses to entrectinib. MAP kinase pathway aberrations occur in childhood cancer including rhabdomyosarcoma and brain tumors. Everolimus is approved for patients with subependymal giant cell astrocytoma. Dabrafenib and vemurafenib have produced durable objective responses in patients with pediatric low-grade glioma harboring BRAF V600E mutations. Selumetinib has been FDA approved for the treatment of plexiform neurofibromas in children with neurofibromatosis type 1 and may have activity in children and adolescents with solid tumors harboring MAP kinase aberrations. Overall, the success of MAP kinase inhibition in children with cancer has been modest, leading to consideration for combinatorial approaches. Crizotinib is approved for the treatment of ALK-positive metastatic NSCLC and has activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and inflammatory myofibroblastic tumors, both associated with ALK fusions. Trials are ongoing to determine the activity of crizotinib and other ALK inhibitors in children with neuroblastoma with known translocations, mutations or amplifications in the ALK gene. Clinical trials of multi-targeted kinase inhibitors including cabozantinib, regorafenib, sorafenib and vandetanib have demonstrated activity in a variety of solid tumors. Clinical trials of these agents have been based on mechanistic rationale and pre-clinical evaluation rather than biomarker selection. Through large scale genomic sequencing efforts in the US and Europe actionable molecular alterations including mutations, amplifications and fusions have been identified in a variety of childhood cancers. Compared to cancers in adults, these molecular aberrations occur less frequently in most cancers in children. However, due to dramatic objective responses and the potential for improved response with decreased toxicity, efforts to evaluate and incorporate targeted agents into multimodality therapy for children with cancer remains a major focus. To date, multi-targeted kinase inhibitors and drugs that target fusion oncoproteins that are histology-agnostic oncogenic drivers in cancers that occur in both child and adults have been most successful. Understanding the characteristics of successful target- drug pairs is critically important to the prioritization of new drugs and clinical trials in children as well as the development of new therapies for pediatric specific targets. Citation Format: Elizabeth Fox. Successes and failures of targeted drug development in pediatric tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr ED01-02. doi:10.1158/1535-7163.TARG-19-ED01-02

Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein

Chromosomal translocation occurs in some cancer cells, resulting in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate expression of the BCR-ABL protein. Recently, we have devised a protein knockdown system by hybrid molecules named Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER). This system is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins. In this review, we describe the development of SNIPER against BCR-ABL, and discuss the features and prospect for treatment of CML.

Annals for Educators - 21 August 2018.

Annals for Educators - 21 August 2018.

Larotrectinib (LOXO-101).

One of the most challenging issues in oncology research and treatment is identifying oncogenic drivers within an individual patient's tumor which can be directly targeted by a clinically available therapeutic drug. In this context, gene fusions as one important example of genetic aberrations leading to carcinogenesis follow the widely accepted concept that cell growth and proliferation are driven by the accomplished fusion (usually involving former proto-oncogenes) and may therefore be successfully inhibited by substances directed against the fusion. This concept has already been established with oncogenic gene fusions like BCR-ABL in chronic myelogenous leukemia (CML) or anaplastic lymphoma kinase (ALK) in lung cancer, including special tyrosine kinase inhibitors (TKIs) which are able to block the activation of the depending downstream proliferation pathways and, consequently, tumor growth. During the last decade, the NTRK1, 2, and 3 genes, encoding the TRKA, B, and C proteins, have attracted increasing attention as another significant and targetable gene fusion in a variety of cancers. Several TRK inhibitors have been developed, and one of them, Larotrectinib (formerly known as LOXO-101), represents an orally available, selective inhibitor of the TRK receptor family that has already shown substantial clinical benefit in both pediatric and adult patients harboring an NTRK gene fusion over the last few years.

Invited Commentary

The study by Li and associates [1Li Y. Yang H.-X. Luo R.-Z. et al.High expression of p300 has an unfavorable impact on survival in resectable esophageal squamous cell carcinoma.Ann Thorac Surg. 2011; 91: 1531-1538Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar] examines the clinical and prognostic significance of p300 expression, a transcriptional regulator, in resectable esophageal squamous cell cancer. Current oncologic research in a variety of malignancies has increasingly focused on identifying genetic fingerprints of tumors, with significant impact on clinical treatment and outcomes. Such examples include EGFR in lung cancer, BCR-ABL in chronic myelogenous leukemia [2Druker B.J. Sawyers C.L. Kantarjian H. et al.Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.N Engl J Med. 2001; 344: 1038-1042Crossref PubMed Scopus (2418) Google Scholar], C-Kit inhibition in gastrointestinal stromal tumors [3Demetri G.D. van Oosterom A.T. Garrett C.R. et al.Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors.N Engl J Med. 2002; 347: 472-480Crossref PubMed Scopus (3658) Google Scholar], K-Ras in colon cancer [4Khambata-Ford S. Garrett C.R. Meropol N.J. et al.Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab.J Clin Oncol. 2007; 25: 3230-3237Crossref PubMed Scopus (1017) Google Scholar, 5Lièvre A. Bachet J.B. Boige V. et al.KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab.J Clin Oncol. 2008; 26: 374-379Crossref PubMed Scopus (1325) Google Scholar], and HER2 blockade in breast cancers [6Slamon D.J. Leyland-Jones B. Shak S. et al.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.N Engl J Med. 2001; 344: 783-792Crossref PubMed Scopus (9166) Google Scholar]. In addition, research is continuing to identify new molecular markers and genetic targets such as B-RAF in melanoma, ALK in lung cancer, and P13K in breast cancer among others. However, specific molecular markers are not yet available for clinical use in esophageal cancer as there are no reliable markers that provide a true clinical assessment of survival and response to treatment. The current study used tissue microarrays to correlate increased expression of the transcriptional co-activator p300, with higher histologic grade, T status, and lymph node involvement in patients with esophageal squamous cell cancer. The p300 transcriptional regulator, also known as KAT3B/EP300, is involved in DNA repair, cell growth and differentiation, apoptosis, and cell migration, with overexpression linked to poor prognosis in lung, breast, colorectal, and prostate cancers. This association with poor outcomes has now been demonstrated in patients with esophageal squamous cell carcinoma with higher p300 expression and is correlated with poorer disease-free and overall survival, particularly in patients with stage II disease. These findings suggest that there may be clinical utility in assessing p300 levels in selected patient populations. Although a serum marker that can identify and prognosticate patient outcomes without a formal biopsy would be the most useful approach, work such as the identification of p300 expression within tumors is important in establishing the first step toward a better understanding of esophageal cancer and in identifying early biomarkers that may be predictive of patient outcomes in this disease. Clearly, further work is needed in esophageal cancer to identify potential treatment targets—as has been done for breast, lung, and stromal tumors—and to extend prognostic biomarkers to include both squamous and adenocarcinoma histologic characteristics. It is the ultimate goal of personalized medicine to use an array of markers to better understand the pathogenesis, tumor targets, recurrence risk, and metastatic potential so that treatment can truly be individualized to the patient and their specific tumor. High Expression of p300 Has an Unfavorable Impact on Survival in Resectable Esophageal Squamous Cell CarcinomaThe Annals of Thoracic SurgeryVol. 91Issue 5Previewp300 is a transcriptional regulator that is involved in fundamental processes such as cell proliferation, cell differentiation, and tumor progression. However, its role and clinical significance in resectable esophageal squamous cell carcinoma (ESCC) has not been elucidated. The purpose of this study was to explore whether there was a correlation between the expression of p300 by immunohistochemistry and the clinical outcome of a group of patients with ESCC treated with surgical resection. Full-Text PDF

As 4 S 4 targets RING-type E3 ligase c-CBL to induce degradation of BCR-ABL in chronic myelogenous leukemia

Arsenic, a curative agent for acute promyelocytic leukemia, induces cell apoptosis and degradation of BCR-ABL in chronic myelogenous leukemia (CML). We demonstrated that ubiquitination and degradation of BCR-ABL was mediated by c-CBL, a RING-type E3 ligase that was also shown to be involved in ubiquitination for many other receptor/protein tyrosine kinases. Our data showed that c-CBL protein was considerably up-regulated by arsenic sulfide (As(4)S(4)). Interestingly, arsenic directly bound the RING finger domain of c-CBL to inhibit its self-ubiquitination/degradation without interfering with the enhancement of ubiquitination and subsequent proteolysis of its substrate BCR-ABL. Degradation of BCR-ABL due to c-CBL induction as a result of arsenic treatment was also observed in vivo in CML mice. These findings provide insight into the molecular mechanisms of arsenic and further support its therapeutic applications in CML in combination with tyrosine kinase inhibitors and potentially also in other malignancies involving aberrant receptor/protein tyrosine kinase signaling.

Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth

The protein signal transducer and activator of transcription 5 (STAT5) of the JAK/STAT pathway is constitutively activated because of its phosphorylation by tyrosine kinase activity of fusion protein BCR-ABL in chronic myelogenous leukemia (CML) cells. This study investigated the potential therapeutic effect of STAT5 decoy oligodeoxynucleotides (ODN) using leukemia K562 cells as a model. Our results showed that transfection of 21-mer-long STAT5 decoy ODN into K562 cells effectively inhibited cell proliferation and induced cell apoptosis. Further, STAT5 decoy ODN downregulated STAT5 targets bcl-xL, cyclinD1, and c-myc at both mRNA and protein levels in a sequence-specific manner. Collectively, these data demonstrate the therapeutic effect of blocking the STAT5 signal pathway by cis-element decoy for cancer characterized by constitutive STAT5 activation. Thus, our study provides support for STAT5 as a potential target downstream of BCR-ABL for CML treatment and helps establish the concept of targeting STAT5 by decoy ODN as a novel therapy approach for imatinib-resistant CML.

Jak2 Phosphorylates Tyr 177 of Bcr-Abl Activating the Ras and PI-3 Kinase Pathways and Maintains Functional Levels of Bcr-Abl in Chronic Myelogenous Leukemia.

Abstract 39We have shown that Jak2 is activated in Bcr-Abl+ hematopoietic cells. Our findings indicate that Jak2 kinase inhibition overcomes drug-resistance in CML cells (Samanta et al., 2006). Recently, we showed that Jak2 inhibition deactivates the Lyn kinase in Bcr-Abl+ cells by inhibiting SET expression; SET inhibits the PP2A-SHP1 (Samanta et al., 2009). Because of the critical effects of Jak2 in Bcr-Abl+ cells, we explored the mechanism of Jak2 action in CML cells. Bcr-Abl is known to be phosphorylated on a number of tyrosine residues including tyrosine 177. Phosphorylation of Tyr 177 leads to binding to Grb2, activation of the SOS-Ras pathway, and activation of the PI-3 kinase through the Grb2-Gab2 pathway. Here, we show that Jak2 and not Bcr-Abl phosphorylates Tyr 177 of Bcr-Abl. Tyr 177 of Bcr-Abl (YVNV) has the Jak2 consensus target sequence (YxxV/L/I) (Argetsinger et al., 2004) as do a number of other Tyr residues in the Bcr portion of Bcr-Abl (b3a2). Inhibition of Jak2 by a specific Jak2 kinase inhibitor TG101209 (TargeGen Inc., San Diego, CA) and a new Jak2 kinase inhibitor (WP1193, synthesized by Dr. Priebe) in Bcr-Abl+ cells, but not inhibition of Bcr-Abl by imatinib mesylate (IM), rapidly reduced phosphorylation of Tyr 177 of Bcr-Abl in IM-sensitive and –resistant cell lines including K562, K562-R, Bcr-Abl+ 32D, Bcr-Abl+ BaF3, Bcr-Abl+T315I BaF3 cells and cells from blast crisis CML patients. Jak2 inhibitors but not IM also strongly inhibited the phosphorylation of a synthetic Bcr peptide containing Tyr 177 (and the surrounding Bcr sequences) catalyzed by either recombinant purified Jak2 or Jak2 immune complexes. Jak2 inhibition of Bcr-Abl+ 32D cells rapidly reduced Grb2 binding to Bcr-Abl, and diminished activation of Ras and PI-3 kinase pathways within two h. Importantly, knockdown of Jak2 with Jak2-specific siRNA also reduced levels of pTyr 177 Bcr-Abl and total Bcr-Abl protein. Rescue experiments reversed Jak2 knockdown and stimulated pTyr 177 levels. In whole cells the total levels of the Bcr-Abl protein also decreased upon Jak2 inhibition with the above chemical inhibitors but at a rate slower than the rate observed in the inhibition of the Tyr 177 phosphorylation of Bcr-Abl. In kinase assays with Bcr-Abl network complexes from Bcr-Abl+ cells, phosphorylation of Tyr 177 of Bcr-Abl was drastically reduced by Jak2 inhibition but Bcr-Abl protein levels remained stable. Reduction of Bcr-Abl protein levels in Bcr-Abl+ cells by Jak2 inhibition also caused a severe reduction of tyrosine phosphorylation of STAT5 and STAT3. We have shown by gel filtration column chromatography of lysates isolated from Bcr-Abl+ cells that Bcr-Abl is physically associated with the IL-3 receptor beta chain, Jak2, Akt and HSP90 in high molecular weight complexes. GSK3β, STAT5 and STAT3 also emerge from the column in the high molecular weight fractions and suggested that several high molecular weight complexes may exist. Nevertheless, incubation of Bcr-Abl+ cells with WP1193 caused disruption of these large molecular network complexes within three h. Nude mice, injected subcutaneously with IM-resistant K562-R cells developed solid tumors, which were reduced in weight by i.p. injection of WP1193 every 48 h at 30 mg/Kg over 10 days. WP1193 at a similar dosage strongly reduced leukemic and tumor effects of Bcr-Abl+ 32D cells expressing T315I Bcr-Abl injected i.v. into nude mice. WP1193 injections into nude mice did not cause significant toxicity over a 10 day period. In summary, Jak2 inhibitors TG101209/WP1193 rapidly reduced levels of pTyr 177 Bcr-Abl, reduced levels of Bcr-Abl protein and induced high levels of apoptosis in IM-sensitive and –resistant Bcr-Abl+ cell lines including T315I cells, CML cell lines, cells from CML patients in blast crisis. In addition Jak2 inhibition reduced tumor formation in mice injected with IM-resistant K562-R and T315I Bcr-Abl+ mouse cells. These findings establish: 1) Jak2 as a major driver of Bcr-Abl signaling pathways in Bcr-Abl+ CML cells, which were previously thought to be activated directly by Bcr-Abl. 2) Although Jak2 is activated by Bcr-Abl, Jak2 is the dominant target molecule in CML and Jak2 becomes even more dominant in CML cells that become resistant to IM and other tyrosine kinase inhibitors. These findings indicate that CML cells, because of the dominant role of Jak2, resemble leukemia cells from Bcr-Abl negative myeloproliferative diseases. Disclosures:No relevant conflicts of interest to declare.

E3 Ligase c-CBL Mediates Ubiquitination-Proteasomal Degradation of BCR-ABL and Therapeutic Effects against BCR-ABL Leukemia.

Abstract 3257Poster Board III-1We previously demonstrated that the cellular level of the fusion oncoprotein BCR-ABL in chronic myelogenous leukemia (CML) could be regulated by the proteolysis via ubiquitin-proteasome pathway and arsenic sulfide (As4S4) could enhance this process to exert therapeutic effects against CML. However, the exact biochemical mechanism and molecules involved, including the ubiquitin E3 ligases of BCR-ABL, their modification sites and the topologic type of ubiquitination remain uncovered. In this work, by using immunoprecipitation (IP) /2D-nano-HPLC/MALDI-TOF-TOF, we found a number of components in ubiquitination process, such as ubiquitin, E1, E2 and E3 ligases in the BCR-ABL protein complex. Particularly, several E3 ligases including the CBL family E3 ligase c-CBL were identified. The in vitro ubiquitination experiments indicated that c-CBL was one of the E3 ligases of BCR-ABL. Overexpression and knockdown experiments of the c-CBL gene revealed it participated in the process of endogeneous BCR-ABL ubiquitination and turnover. The enforced overexpression of c-CBL could enhance the degradation of BCR-ABL and apoptosis of CML cells whereas the down regulation of c-CBL generated opposite effects. Furthermore, we mapped a domain of c-CBL as the interface to form complex with BCR-ABL based on the protein structure prediction. Either the identification of BCR-ABL ubiquitination sites or the interaction experiment among the c-CBL and BCR-ABL are undergoing. Our data thus suggest that induction of c-CBL may be a new therapeutic approach for CML. Disclosures:No relevant conflicts of interest to declare.

NADPH Oxidases Are Important Regulators of Growth and Migration in Myeloid Neoplasms.

Abstract 2190Poster Board II-167Myeloid neoplasms are frequently associated with transforming tyrosine kinase oncogenes, including BCR-ABL in chronic myelogenous leukemia (CML), JAK2V617F in myeloproliferative disorders and FLT3.ITD in acute myeloid leukemia (AML). Cells transformed by tyrosine kinase oncogenes have been found to display increased levels of intracellular reactive oxygen species (ROS). Elevated ROS levels have been implicated in genomic instability through oxidative DNA damage, but also regulate cellular functions through redox-sensitive proteins. The majority of intracellular ROS in non-phagocytic cells are produced within the mitochondria. Nevertheless, recent studies indicate a role for NADPH oxidases (NOX) in transformation. There are five NOX genes (NOX1-5) and two genes encoding the related dual oxidases (DUOX1-2) in humans. We asked whether NOXs alter cell growth, migration and ROS production in hematologic malignancies transformed by tyrosine kinase oncogenes. Real-time PCR analysis in patient-derived cell lines KU812 (BCR-ABL), HEL (JAK2V617F) and Molm13 (FLT3.ITD), showed expression of NOX2, NOX4, and NOX5. Murine cells are readily transformed by BCR-ABL, JAK2V617F and FLT3.ITD, even though the gene for NOX5 is absent in mice. Therefore, we targeted NOX2 and NOX4 in KU812, HEL and Molm13 cells using lentiviral-based shRNAs. In addition, we also performed knockdown of p22phox, a common subunit that is required for stability and functioning of NOX1-4, thus also controlling for functional redundancy between the NOX genes. Interestingly, we found that p22phox knockdown reduced cell growth by 16.0-32.0% in KU812, 27.3-50.9% in HEL, and 31.3-54.2% in Molm13 cells (p<0.05, n=3). Similar to cell growth, a drastic reduction in spontaneous migration compared to control cells was observed in KU812 (74.3-87.6%, p<0.05) HEL (47.1-67.6%, p<0.05) and Molm13 (51.0-75.6%, p<0.05) cells with p22phox knockdown. Surprisingly, knockdown of NOX2, NOX4, or p22phox did not significantly reduce intracellular ROS levels. Further, mitochondrial electron transport chain inhibitors led to a decrease in intracellular ROS levels and oxygen consumption in KU812, HEL and Molm13 cells. Using patient samples, a similar decrease in ROS was observed in response to complex III inhibitors myxothiazol (CML: 57.2±9.4%; polycythemia vera: 36.2±0.4%; AML: 51.8±5.2%; p<0.05, n=4) and stigmatellin (CML: 62.5±7.8%; PV: 48.1±4.2%; AML: 53.9±5.5%; p<0.05, n=4). Consistent with the above data, KU812, HEL and Molm13 cells treated with their respective tyrosine kinase inhibitors, showed a reduction in MitoSOX Red fluorescence, an indication for reduced mitochondrial superoxide radicals (KU812: -38.9%; HEL: -31.3%; Molm13: -41.9%), further implicating mitochondria in the excess generation of intracellular ROS. Overall, these data suggest that expression of NOX proteins is required for optimal growth and migration of myeloid leukemia cells and therefore hints at novel targets for drug development that may aid traditional therapy. (supported in part by NIH/NCI grant 5R01CA134660-02) Disclosures:No relevant conflicts of interest to declare.

Chronic myelogenous leukemia stem cells: What’s new?

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that arises in the hematopoietic stem cell compartment. CML is one of the best-understood malignancies, as it results from a single genetic mutation, the fusion oncogene BCR-ABL, which has been widely studied. Specific tyrosine kinase inhibitors have been developed to target BCR-ABL in CML, but these agents fail to eliminate the CML stem cell population and thus are unlikely to cure CML. This article reviews recent developments in the biology and treatment of CML, specifically focusing on CML stem cells. Significant progress continues to be made in our understanding of CML stem cell biology, which has wider implications within the cancer stem cell field. We are also beginning to see the identification of novel therapies that specifically target the CML stem cell. These are exciting times in the quest to cure CML.

Oncogenic Signals as Treatment Targets in Classic Myeloproliferative Neoplasms

Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPNs) include BCR-ABL in chronic myelogenous leukemia (CML) and a spectrum of PDGFRA/B mutant proteins that are products of intra- (eg, FIP1L1-PDGFRA) or interchromosomal (eg, ETV6-PDGFRB) gene fusions. Other MPN-relevant putative oncogenes that are awaiting therapeutic validation, include JAK2 and MPL mutations in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF); KITD816V and other KIT mutations in systemic mastocytosis, and FGFR1 rearrangements associated with the 8p11 leukemia/lymphoma syndrome. The current review focuses on mutant molecules of interest in classic MPNs (ie, CML, PV, ET, and PMF) in the context of their value as drug targets.

Quantitative realtime RTPCR monitoring of BCR-ABL in chronic myelogenous leukemia shows lack of agreement in blood and bone marrow samples

Quantitative realtime RTPCR monitoring of BCR-ABL in chronic myelogenous leukemia shows lack of agreement in blood and bone marrow samples

Janus Kinase (JAK)-2 Does Not Play a Role in Bcr-Abl Signaling in Philadelphia Chromosome (Ph)-Positive (p190) Acute Lymphoblastic Leukemia (ALL) Cells.

Novel immunochemotherapy regimens combined with imatinib mesylate (IA) have significantly improved treatment outcome of Ph+ ALL. Nevertheless, most adult patients with Ph+ ALL relapse and succumb to their disease. Recent reports suggested that Jak-2 is engaged in the signaling of Bcr-Abl in chronic myelogenous leukemia (CML) cells. Because Jak-2 inhibitory agents are currently investigated in clinical trials, we sought to explore the role of Jak-2 in the signaling of Bcr-Abl in Ph+ ALL assuming that inhibition of Jak-2 might be beneficial in the treatment of Ph+ ALL. To do this, we used our Ph+ (p190) ALL cell lines Z-119 and Z-181 (Estrov et al. J Cell Physiol 166: 618, 1996). We chose these cells because in both lines Jak-2 can be activated. Both Z-119 and Z-181 cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors and GM-CSF activates Jak-2 and stimulates the proliferation of both cell lines. Using a clonogenic assay, we found that IA inhibited the proliferation of these cells at concentrations ranging from 50 to 500 nM. Because Bcr-Abl was found to activate the signal transducer and activator of transcription (STAT)-5 in CML cells, we used Western immunoblotting and found that IA inhibited the phosphorylation (p) of STAT5 in a dose-dependent manner in Ph+ ALL cells. To test whether JAk-2 plays a role in Bcr-Abl (p190) signaling we incubated Z-181 cells for 4 hours with or without 50, 100, 250, and 500 nM IA, extracted cellular protein and immunoprecipitated total STAT5 protein. Then, using Western immunoblotting we detected the Bcr-Abl p190 protein in all STAT5 immunoprecipitates and by using specific pSTAT5 antibodies, we demonstrated that IA induced a dose-dependent reduction in the levels of pSTAT5, but not of p190 protein, suggesting that the p190 Bcr-Abl kinase binds to and activates STAT5. Remarkably, neither Jak-2 nor pJak-2 was detected in either immunoprecipitate. To further delineate the role of Jak-2 in Bcr-Abl signaling we extracted protein from Z-181 cells and immunoprecipitated Jak-2. Neither Bcr-Abl nor STAT5 was detected in these immunoprecipitates, confirming that Jak-2 does not bind Bcr-Abl p190 protein and does not participate in the activation of STAT5. Taken together, our data suggest that Bcr-Abl (p190) binds and phosphorylates STAT5 whereas, Jak-2 is not engaged in Bcr-Abl (p190) signaling in Ph+ ALL cells.

Nilotinib therapy in chronic myelogenous leukemia

Imatinib mesylate (Gleevec; Novartis, Basel, Switzerland) is a highly effective inhibitor of the deregulated kinase activity of BCR-ABL in chronic myelogenous leukemia (CML) and represents the current standard of care for patients with this disease. Mutations within the ABL kinase domain that interfere with drug binding have been identified as the main mechanism of resistance to imatinib. Currently, more than 50 different BCR-ABL mutants conferring varying degrees of resistance to tyrosine kinase inhibitors have been identified. Nilotinib (Tasigna; Novartis) is a second-generation tyrosine kinase inhibitor with 30-fold higher potency against BCR-ABL kinase than imatinib. Notably, nilotinib is active against a wide range of imatinib-resistant or-intolerant patients, except for T315I. Results from the pivotal phase II studies of nilotinib for patients with CML after failure or intolerance to imatinib therapy indicate that nilotinib has a favorable toxicity profile and is highly efficacious in this setting. Studies exploring the efficacy of nilotinib as front-line therapy for patients with newly diagnosed CML are ongoing. Here, we review the preclinical and clinical development of nilotinib for the treatment of CML.

Quantitative real-time RT-PCR monitoring of BCR-ABL in chronic myelogenous leukemia shows lack of agreement in blood and bone marrow samples

Molecular monitoring of the BCR-ABL transcript in chronic myelogenous leukemia (CML) using quantitative RT-PCR provides clinicians with important diagnostic and prognostic information. To determine whether molecular detection and monitoring of CML is comparable using peripheral blood (PB) and bone marrow (BM) aspirate samples, we performed a prospective study using quantitative real-time RT-PCR (QRT-PCR) of paired PB and BM samples from 41 patients with CML entered onto a single Cancer and Leukemia Group B (CALGB) treatment study. QRT-PCR analysis of PB and BM samples was performed prior to initiation of, and during, treatment with homoharringtonine and cytarabine on a CALGB study for previously untreated CML. Statistical analyses demonstrated good agreement of PB and BM pre-treatment samples. However, using the Bland-Altman statistical method that measures true agreement between PB and BM values, we found that there was only modest agreement of BCR-ABL measurements in PB and BM for samples obtained during treatment. PB values obtained during treatment tended to be lower than the corresponding BM values [average difference = -0.37 (p<0.001) in 36 paired samples] and the 95% limits of agreement ranged from -1.23 to 0.48. Nevertheless, our study demonstrates that BM and PB QRT-PCR values followed a similar trend during treatment (Spearman correlation coefficient, 0.83; 95% CI, 0.70, 0.96). Our data suggest that, quantitatively, PB and BM measurements of BCR-ABL are frequently disparate. Since BM values tended to be higher than PB values, BM sampling provides the most accurate assessment of minimal residual disease (MRD). Based on these results, we caution against interchanging BM with PB sampling for MRD monitoring during treatment of CML since this may lead to misinterpretation of treatment results.