Addition Of Dasatinib Research Articles

Trametinib alters contractility of paediatric Noonan syndrome-associated hypertrophic myocardial tissue slices.

No curative treatment is available for RASopathy-associated childhood-onset hypertrophic cardiomyopathy (RAS-CM). Preclinical data and individual reports suggest a beneficial effect of small molecules targeting the RAS-mitogen-activated protein (MAP) kinase (MAPK) pathway in severely affected RAS-CM patients. The aim of this study was to evaluate the biophysical effects of trametinib, rapamycin and dasatinib on cultivated myocardial tissue slices of a paediatric RAS-CM patient using biomimetic cultivation chambers (BMCCs) and to correlate the findings with clinical data. Contracting right ventricular (RV) tissue slices were prepared from resected myocardium, cultivated in BMCCs and treated with distinct molecules directly and indirectly targeting the RAS-MAPK pathway (trametinib, rapamycin and dasatinib) or dimethyl sulfoxide (DMSO). Tissue biophysical properties were assessed using electrical stimulation protocols. Contractile function, force-frequency relationship and post-pause potentiation were compared before and after treatment. These parameters correlated to L-type Ca2+ channel function and sarcoplasmic Ca2+ loading. In vivo, off-label treatment with MAPK kinase (MEK) inhibitor trametinib of a child with severe RAS-CM resulted in a modest reduction of RV outflow tract (RVOT) obstruction (RVOT 151 to 122mmHg after 11weeks) and improved diastolic function (E/A 0.68 to 1.09 after 11weeks) and myocardial strain [RV global radial strain (RV-GRS) 25.94 to 42.76; RV global circumferential strain (RV-GCS) -15.26 to -18.61; and RV global longitudinal strain (RV-GLS) -10.31 to -16.78 at 11weeks], as determined by echocardiography and cardiac magnetic resonance tomography. In cultivated RV myocardial tissue slices, contraction force decreased after addition of trametinib and rapamycin but not after addition of DMSO and dasatinib. Improvement of Ca2+ handling, as depicted by a more positive force-frequency relationship and enhanced post-pause potentiation (31.2%), was noted in the trametinib-treated slice. The increase in post-pause potentiation was less pronounced in rapamycin-treated (26%) and absent in dasatinib-treated (<1%) slices. Ex vivo analysis of cultivated and electrically stimulated RV myocardial tissue slices of a patient with RAS-CM showed decreased contractility and improved sarcoplasmic reticulum function after addition of trametinib and in part after addition of rapamycin, but not after addition of dasatinib.

Philadelphia chromosome-like acute lymphoblastic leukemia with concomitant rearrangements of CRLF2 and ABL1: a pediatric case report

BackgroundBCR::ABL1-like or Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) was first reported in 2009. Ph-like ALL is characterized by gene signature similar to Philadelphia chromosome ALL, but without BCR::ABL1 fusions. Molecularly, Ph-like ALL is divided into seven categories, with CRLF2 and ABL-class rearrangements being the two most common subtypes, exhibiting alterations in distinct downstream signaling cascades.Case presentationWe report a rare case of pediatric Ph-like ALL with concomitant CRLF2 and ABL1 rearrangements. CRLF2 was fused with P2RY8, its most common fusion partner, whereas ABL1 was fused with MYO18B, a novel fusion partner that has not been previously reported. The 4-year-old female patient was treated using the national multicenter CCCG-ALL-2020 protocol with the addition of dasatinib at the end of induction when ABL1 rearrangement was confirmed by RNA-seq. Morphologically and molecularly, the patient remained in continuous remission until the last follow-up. To the best of our knowledge, this is the first case of Ph-like ALL harboring two distinct rearrangement categories.ConclusionsOur results identified that ABL1 rearrangement and CRLF2 rearrangement can coexist. The application of FISH, whole transcription sequencing, PCR can help us to have a more comprehensive understanding of ALL cytogenetics and molecular biology. Further studies are needed to explore the role of targeted therapies in such rare clinical scenarios.

Dasatinib overcomes glucocorticoid resistance in B-cell acute lymphoblastic leukemia

Resistance to glucocorticoids (GC) is associated with an increased risk of relapse in B-cell progenitor acute lymphoblastic leukemia (BCP-ALL). Performing transcriptomic and single-cell proteomic studies in healthy B-cell progenitors, we herein identify coordination between the glucocorticoid receptor pathway with B-cell developmental pathways. Healthy pro-B cells most highly express the glucocorticoid receptor, and this developmental expression is conserved in primary BCP-ALL cells from patients at diagnosis and relapse. In-vitro and in vivo glucocorticoid treatment of primary BCP-ALL cells demonstrate that the interplay between B-cell development and the glucocorticoid pathways is crucial for GC resistance in leukemic cells. Gene set enrichment analysis in BCP-ALL cell lines surviving GC treatment show enrichment of B cell receptor signaling pathways. In addition, primary BCP-ALL cells surviving GC treatment in vitro and in vivo demonstrate a late pre-B cell phenotype with activation of PI3K/mTOR and CREB signaling. Dasatinib, a multi-kinase inhibitor, most effectively targets this active signaling in GC-resistant cells, and when combined with glucocorticoids, results in increased cell death in vitro and decreased leukemic burden and prolonged survival in an in vivo xenograft model. Targeting the active signaling through the addition of dasatinib may represent a therapeutic approach to overcome GC resistance in BCP-ALL.

Abstract 3333: Pre-clinical investigation of neratinib plus dasatinib in breast cancer

Abstract Background: HER2-targeted therapies have revolutionized the prognosis of HER2-positive (HER2+) breast cancer (BC). However, the emergence of acquired resistance or de novo resistance are persistent clinical problems. The objective of this study was to examine neratinib (NER), an irreversible pan-HER inhibitor approved for early-stage and metastatic HER2+ BC, and dasatinib (DAS), a multi-kinase inhibitor used to treat certain leukemias, as a new treatment for HER2-targeted therapy resistant HER2+ BC and triple negative BC (TNBC). Methods: The anti-proliferative effect of NER plus DAS was screened in a panel of 20 breast cancer cell lines using a 5-day acid phosphatase assay. This panel included 12 HER2+ (3 trastuzumab (TRAS) sensitive (SKBR3, BT474, EFM192A), 2 acquired TRAS resistant (BT474-T, SKBR3-T), 3 innately TRAS resistant (HCC1954, HCC1569, JIMT1), and 4 acquired neratinib resistant cell lines (HCC1954-N, HCC1569-N, SKBR3-N, BT474-N), 6 triple negative (BT20, HCC1143, HCC1937, MDA-MB- 157/231/468), and 2 estrogen receptor positive (ER+) cell lines (MCF7, ZR-75-1). Drug synergy was examined using Combenefit and Calcusyn software. Combination Index (CI) values &amp;lt; 1 indicated synergy, &amp;gt; 1 antagonism. Neratinib IC50 values &amp;lt; 150nM were considered physiologically relevant. Prolonged exposure in vitro efficacy was tested in these cell lines by treating twice weekly with NER +/- DAS until resistance emerged. Cell confluence was then quantified using a crystal violet-based method. The safety and efficacy of NER (10 mg/kg) plus DAS (15 mg/kg) was investigated in vivo in HER2+ HCC1954 xenografts in BALB/c nude mice. Results: NER was highly effective against HER2+ BC cell lines regardless of TRAS sensitivity (IC50 values range 1-172 nM), showed moderate activity in TNBC (IC50 value range 31 nM - &amp;gt;1 μM), and did not achieve &amp;lt;1 μM activity in ER+ BC cell lines. NER resistant cell lines had IC50 values greater than 150 nM DAS alone displayed IC50 values from 8-135 nM in 4/6 TNBC cell lines, with all 6 TNBC cell lines sensitive to either the single agents or the combination at concentrations less than 100 nM. DAS enhanced the anti-proliferative activity of NER in the HCC1954, HCC1569, JIMT1 cell lines (CI values = 0.27-0.62). The NER/DAS combination overcame NER resistance in all acquired NER-resistant cell line models and no antagonism was observed in any cell line tested. Matrix assays confirmed synergy at low nanomolar concentrations of both drugs. NER plus DAS had a greater long term in vitro anti-proliferative effect over NER in HCC1954, HCC1569, and JIMT1 cells, preventing or delaying the development of NER resistance. NER plus DAS was well tolerated in mice for over 60 days. The combination was better than single agents and showed prolonged anti-tumor effect against HCC1954 xenografts in vivo (p=0.02). Conclusions: This study suggests that the addition of DAS to NER may have potential as a new treatment strategy for BC, particularly for HER2+ BC. Citation Format: Neil T. Conlon, Sandra Roche, Fiona O'Neill, Justine Meiller, John Crown, Denis M. Collins. Pre-clinical investigation of neratinib plus dasatinib in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3333.

How I treat chronic-phase chronic myelogenous leukemia

AbstractWhen imatinib, the first tyrosine kinase inhibitor (TKI) developed for use in chronic myelogenous leukemia (CML), was approved in 2001, the treatment of this disease was forever changed. Significant reductions in the molecular burden of disease were seen with the first-generation TKI imatinib and, with the addition of dasatinib (2006), nilotinib (2007), bosutinib (2012), and ponatinib (2013), deeper and more rapid reductions were noted. Physicians could begin to tailor TKI therapy to individual patients, and patients who did not respond to or could not tolerate first-line therapy now had options. Importantly, the number of patients who developed accelerated or blast phase disease decreased dramatically. Research in CML continues to evolve; by presenting illustrative cases, this article reviews some of the newer aspects of clinical care in this disease. Updated information regarding bosutinib and asciminib, the latter currently in clinical trials, will be presented; bosutinib is of particular interest as the drug's transit through the United States Food and Drug Administration highlights the question of what is considered optimal response to TKI therapy. The challenge of understanding the cardiac safety data of ponatinib and the unique dosing schedule based on individual response will be discussed. Lastly, two cases will focus on features of TKI treatment that, remarkably, have become part of the treatment algorithm: family planning for women with CML and stopping therapy after meeting a specific treatment milestone.

Concomitant use of a dual Src/ABL kinase inhibitor eliminates the in vitro efficacy of blinatumomab against Ph+ ALL

AbstractBlinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome–positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Abstract 1773: Targeting HER2 and Src in HER2-driven ovarian and esophagogastric cancers

Abstract Background: Neratinib, an oral, irreversible pan-HER tyrosine kinase inhibitor, has exhibited clinical activity against HER2-mutant and HER2 over-expressing cancers. Our previous pre-clinical studies have shown that combining the multi-kinase inhibitor dasatinib with neratinib is highly synergistic in HER2-positive breast cancer. Therefore, the aim of this study was to examine the potential of combining neratinib and dasatinib in HER2 over-expressing/mutant esophagogastric (EG) and ovarian cancer. Methods: The prevalence of HER2 mutation and amplification levels in ovarian and EG cancers was determined using the AACR Project GENIE database. The anti-proliferative effects of neratinib and dasatinib were assessed by acid phosphatase assay in a panel of HER2-altered ovarian (Caov3, OVCAR8, IGROV-1) and EG (NCI-N87, OE19) cancers. Synergy was assessed using Combenefit software with the Loewe additivity model, with additivity and synergy &amp;gt; 0 and antagonism &amp;lt; 0. Western blotting was used to examine the basal expression of HER2 and downstream signalling activity and to investigate the signalling effects of drug treatment in HER2-mutant OVCAR8 cells. Results: HER2 was the most commonly amplified gene in EG cancers (14.1%) and was also amplified in 2.4% of ovarian cancers. HER2 mutations occurred in 6.1% of EG and 1.5% ovarian cancers. All five cell lines tested displayed nanomolar sensitivity to single agent neratinib or dasatinib (Table). The combination of neratinib and dasatinib was synergistic in all cell lines. The greatest synergy was observed with low concentrations of neratinib in EG cell lines. Synergistic interactions occurred across a range of concentrations with both drugs in ovarian cancer cell lines. Antagonism was not observed at any concentration tested. Neratinib plus dasatinib inhibited Src and MAPK activity in OVCAR8 cells. Neratinib and dasatinib in ovarian and EG cancersCell linesCancertypeNeratinib EC50 value (nM)Dasatinib EC50 value (nM)Combination effectNCI-N87Esophagogastric0.188979SynergismOE19Esophagogastric0.432284SynergismOVCAR8Ovarian16281SynergismIGROV-1Ovarian1706SynergismCaov3Ovarian13730Synergism Conclusions: These data suggest that the addition of dasatinib may enhance the anti-cancer effect of neratinib in HER2-driven ovarian and EG cancers and warrants further investigation. Citation Format: Neil T. Conlon, Keshia Kroh, John Crown, Denis M. Collins. Targeting HER2 and Src in HER2-driven ovarian and esophagogastric cancers [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 1773.

Abstract 126: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in PTEN-null TNBC model

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); but not all patients respond to this combination. The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors. In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in PTEN-null TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The PTEN-null TNBC cell line, MDA-MB-468, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Interestingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation of MDA-MB-468 cells. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further increased the activation of caspase 3 and PARP, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an MDA-MB-468 xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusion: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in PTEN-null TNBC. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in PTEN-null TNBC model [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 126.

Abstract P3-11-19: Preclinical model of TNBC: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination. The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors. In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The BRCA-mutated and PTEN null TNBC cell line, SUM149PT, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an SUM149PT xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusion: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in BRCA-mutated and PTEN null TNBC. Citation Format: yuliang Sun, Xiaoqian Lin, Jennifer Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Preclinical model of TNBC: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-19.

Spectral and computational attributes: Binding of a potent anticancer agent, dasatinib to a transport protein

The present study describes the insights in to the mechanism of interaction between a model protein and an anticancer drug, dasatinib (DAS). DAS is a multi-target anticancer drug which controls the proliferation of mutant expressing cells. Human serum albumin (HSA) is the ubiquitous protein of blood plasma (40–50 g/L) and able to bind a wide range of exogenous and endogenous ligands. Binding mechanism of DAS to HSA was studied under physiological conditions by multi-spectroscopic techniques viz., steady state fluorescence, time resolved fluorescence, fluorescence (Fӧrster) resonance energy transfer (FRET), UV–visible and FTIR besides molecular modeling studies. Fluorescence measurements showed that the fluorescence intensity of HSA was quenched significantly by DAS with a red shift (from 342 to 356 nm) in its emission wavelength. The values of binding constant, number of binding sites and Stern-Volmer quenching constant (Ksv) were calculated for DAS-HSA interactions at different temperatures. Decreased Ksv values with increase in temperature suggested the presence of static quenching mechanism. This was also evident from time resolved fluorescence studies. The probable binding site for DAS in the hydrophobic pocket (sub-domain IIA) of HSA was revealed by both site probe experiments and molecular docking studies. Negative values of ΔG° revealed the spontaneity of DAS-HSA interaction while negative ΔH° and ΔS° values indicated the participation of hydrogen bonding and van der Waals forces in the stabilization of DAS-HSA complex. Further, conformational change in the protein backbone upon the addition of DAS was evident from absorption, FT-IR and synchronous fluorescence spectral results. The effect of essential metal ions on the binding of DAS to protein was also examined.

Abstract 3448: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination (Rugo HS et al. The New England Journal of Medicine. 2016). The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors (Finn RS et al. Clinical Cancer Research. 2011). In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The KRAS/BRAF-mutated TNBC cell line, MDA-MB-231, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an MDA-MB-231 xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusions: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in patients with KRAS/BRAF-mutated TNBC. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer C. Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in a preclinical model of TNBC [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 3448.

Combination Chemotherapy Plus Dasatinib Leads to Comparable Rates of Overall Survival and Relapse-Free Survival As Allogeneic Hematopoietic Stem Cell Transplantation in Philadelphia Positive Acute Lymphoblastic Leukemia

Introduction:The presence of the Philadelphia chromosome has historically been associated with a poor prognosis in patients with acute lymphoblastic leukemia (ALL). Prior studies had demonstrated that the 5-year overall survival (OS) rate among adult patients with Philadelphia-chromosome positive (Ph+) ALL was 25% compared to 41% in Philadelphia-chromosome negative (Ph-) ALL (Moorman et. al. Blood 2007). While hematopoietic stem cell transplantation (HSCT) has long been regarded as the standard of care for adult Ph+ ALL patients despite its significant transplant-related morbidity and mortality, its benefit is less clear in the era of newer-generation tyrosine kinase inhibitors (TKI) such as dasatinib.Methods:We conducted a retrospective study at a single urban transplant center with academic affiliation to analyze the outcomes of adult patients diagnosed with Ph+ ALL between 2005-2018. Patients were treated with either combination chemotherapy plus dasatinib or combination chemotherapy plus dasatinib and allogeneic HSCT. Chemotherapy regimens included hyper-CVAD, Berlin-Frankfurt-Munster-like (BFM-like) protocol, and a regimen developed at the University of Southern California (USC ALL regimen) using pegaspargase (Douer et. al. Journal of Clinical Oncology 2014). All patients in both the transplant group and the non-transplant group received TKI therapy with dasatinib throughout their treatment courses; in the former, patients received dasatinib both prior to and following their transplants. Statistical analysis was performed using the Fisher Exact Probability Test with p-values <0.05 deemed significant.Results:A total of 71 Ph+ ALL patients were included in this study. Forty-three patients (60.5%) were male and the median age at the time of diagnosis was 44 years (range 20-69). Median follow-up time was 15 months (range 1-131). Chemotherapy regimens included the USC ALL regimen in 69.8% of patients, hyper-CVAD in 25.6%, and BFM-like protocol in 7%. While in remission, 31 (43.6%) patients underwent allogeneic HSCT with subsequent post-transplant dasatinib therapy. Median time of dasatinib initiation following transplant was at day 102. Matched related donor transplants were performed in 48% of patients, matched unrelated donor transplants in 24%, and haploidentical donor transplants in 29% of patients. In comparing OS rates, 1-year OS was 92.9% vs. 100% (p = 0.49), 2-year OS was 90.1% vs. 83.3% (p = 0.60), and 3-year OS was 75% vs. 62.5% (p = 0.64) in the allogeneic HSCT group compared to the non-transplant group, respectively (Figure 1). The 3-year relapse-free survival (RFS) rates also proved to be similar at 70.5% in the allogeneic HSCT group and 80.1% in the non-transplant group (p = 0.94, Figure 2). In addition, 37% of patients had a poor prognostic factor of a white blood count (WBC) >30 x 109/L at the time of diagnosis. When comparing rates of OS and RFS between transplant vs. non-transplant patients in this group, there was also no significant difference.Conclusion:This study showed that in the management of adult Ph+ ALL patients, there was no significant difference in OS or RFS between patients who underwent allogeneic HSCT compared to those who received only combination chemotherapy with dasatinib. In addition, prior studies of Ph+ ALL patients treated with either chemotherapy alone or chemotherapy followed by HSCT concluded that the OS rates were approximately 50%, 35%, and 30% at the 1-year, 2-year, and 3-year marks, respectively (Rowe et. al. Blood 2005). Our analysis, however, demonstrated improved rates of survival at all time points for this patient population with the addition of dasatinib. Subgroup analysis of patients with a WBC >30 x 109/L at the time of diagnosis also showed no significant difference in OS between transplant and non-transplant patients despite previous reports showing a survival benefit from HSCT (Huang et. al. Blood 2016). This may be attributed to the fact that our patients received the newer and more potent agent dasatinib compared to imatinib in these prior studies. Since allogeneic HSCT demonstrated no survival benefit in our study and can also introduce risks of serious infectious complications, venoocculsive disease (VOD), and graft-versus-host disease (GVHD) that contribute to patient morbidity and mortality, it may serve a less beneficial role for the Ph+ ALL population in the era of newer-generation TKIs. DisclosuresNo relevant conflicts of interest to declare.

Abstract P4-03-15: Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer

Abstract Background: Afatinib is an irreversible pan-HER inhibitor approved for non-small cell lung cancer. We have previously shown that afatinib inhibits growth of HER2-positive breast cancer cells and enhances response to trastuzumab. However, we have also shown that long-term exposure to tyrosine kinase inhibitors leads to the development of acquired resistance. To determine if acquired afatinib resistance develops in HER2-positive breast cancer cells, we exposed a HER2-positive breast cancer cell line to afatinib for 6 months and investigated alterations in the cells following long-term exposure. Methods: SKBR3 cells were treated with 150 nM afatinib twice-weekly for 6 months. Growth response to drug inhibitors was assessed by acid phosphatase assay. Drug sensitivity was examined in four HER2-positive cell lines (SKBR3, EFM192A, BT474 and HCC1954) and three acquired trastuzumab resistant cell lines (SKBR3-T, BT474-T and EFM192A-T). Reverse phase protein array (RPPA) was used to determine alterations in key signaling pathways. Src, p-Src, EGFR, p-EGFR, ERK1/2, p-ERK 1/2 levels were examined by Western blotting. To examine the prevention of the development of afatinib resistance, cells were treated twice weekly with afatinib, dasatinib, or the combination and stained with crystal violet when confluent. Results: Following 6 months of afatinib treatment, the SKBR3-A cells were more resistant to afatinib compared to parental cells (IC50 SKBR3-A 284 ± 28.2 nM vs SKBR3-Par 10.9 ± 3.4 nM). Furthermore, the resistant cells were cross-resistant to lapatinib, neratinib and trastuzumab. RPPA interrogation of the SKBR3-A cells showed alterations in several pathways, including significantly increased levels of p-Src (Y416). SKBR3-A cells were more sensitive to Src inhibition with dasatinib compared to SKBR3-Par cells and the combination of afatinib and dasatinib was highly synergistic in SKBR3-A cells (CI value = 0.09 ± 0.06). The combination of afatinib and dasatinib was also synergistic in the trastuzumab resistant SKBR3-T cells (Table). Afatinib and dasatinib inhibited EGFR and Src activation and ERK 1/2 signalling in SKBR3-A cells. Short-term resistance assays showed that the addition of dasatinib to afatinib blocks the emergence of resistant cells in three of four HER2 positive cell lines tested and two of the three acquired trastuzumab resistant cell lines tetsed. Conclusion: HER2-positive breast cancer cells that are highly sensitive to afatinib can develop acquired resistance to afatinib within six months. Src is a potential target to prevent the development of afatinib resistance and thus combined treatment with afatinib and dasatinib may be beneficial in patients with HER2-positive breast cancer. Percentage growth in HER2-positive cells treated with afatinib and/or dasatinib for 5 days. 20 nM Afatinib40 nM Dasatinib20 nM Afatinib + 40 nM DasatinibSKBR325.2 ± 6.1109.9 ± 6.222.6 ± 6.2SKBR3-A62.1 ± 0.787.3 ± 11.036.3 ± 0.3*SKBR3-T42.6 ± 4.9107.4 ± 9.729.3 ± 3.5*BT47418.2 ± 3.079.5 ± 5.213.3 ± 1.1BT474-T6.9 ± 2.795.3 ± 2.06.2 ± 2.3EFM192A39.1 ± 4.7105.7 ± 3.135.3 ± 2.7EFM192A-T30.4 ± 5.299.8 ± 2.528.5 ± 4.4HCC195461.8 ± 8.180.5 ± 8.219.4 ± 3.6** indicates enhanced anti-proliferative response for the combination compared to the single agents. Citation Format: Conlon N, Canonici A, Morgan C, Cremona M, Hennessey BT, Eustace A, O'Brien N, Slamon D, Crown J, O'Donovan N. Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-15.

Comprehensive Genomic Profiling for Improved Diagnosis and Therapy of Pediatric Acute Leukemias

Subsets of pediatric acute leukemias are refractory to frontline therapy or relapse with standard treatment. Pediatric leukemias are associated with distinct molecular and genomic alterations. Clinical genomic profiling has been used effectively to improve the diagnosis of adult leukemias. However, its use and utility for pediatric leukemias are not well defined. Here, we used FoundationOne Heme, a clinical grade, high-throughput, hybridization capture-based next-generation sequencing (NGS) assay for targeted sequencing of all exons of 405 genes as well as RNA sequencing of 265 genes. We analyzed the results of samples from 71 patients sequenced on the FoundationOne® Heme assay from children and young adults with acute lymphoblastic leukemia (ALL) (n= 34); early T cell precursor ALL (ETP) (n=3); acute myelogenous leukemia (AML) (n= 19); ambiguous lineage leukemia (n=3); and myelodysplastic syndrome (MDS) (n=12) who were treated at Memorial Sloan Kettering Cancer Center in the Department of Pediatrics over a 26 month period (January 2014-March2016). Age of patients ranged from 1-24 years of age (median =10 years). 42 samples were from patients who had yet to receive cytotoxic therapy (newly diagnosed) and 29 samples were from patients who had relapsed (n=24) or had refractory disease (n=5).The current average turnaround time for FoundationOne® Heme is 12-14 days. Overall 99% (70/71) of specimens were successfully profiled. We identified 230 genomic alterations, including 59.1% (136/230) missense or nonsense mutations of genes, 15.6% (36/230) copy number alterations, and 25.6% (59/230) gene fusions or rearrangements. Molecular aberrations were detected in 81% (9/11) of patients with normal karyotypes; 55% (5/9) of these were cytogenetically cryptic genomic rearrangements including NUP98-NSD1 (n=2) and 1 MLL-PTD (n=1). We identified one novel translocation involving SATB1-PDGFRB in a sample from a patient with pre B ALL. Another patient with pre B ALL was found to have a unique combination of genomic alterations including an MLL-PTD and SSBP2-JAK2. Two patients with MDS were identified as harboring germline GATA2 mutations, indicating the likely benefit of an allogeneic hematopoietic stem cell transplantation. Three patients had therapy modifications based on findings with the addition of dasatinib (ZMIZ1-ABL1), ruxolitinib (PCM1-JAK2) and trametinib (PTPN11mutation), and two patients had risk stratification altered based on MLL rearrangements which were not identifiable by conventional cytogenetics.Overall, the use of comprehensive genomic profiling led to improved accuracy of diagnosis or alteration of therapy in 10% (7/71) cases, including molecular based therapy selection (n=3), escalation of conventional chemotherapy due to high-risk features (n=2), and inclusion of stem cell transplantation (n=2). Thus, clinical genomic profiling of pediatric acute leukemia led to discovery of new pathobiology, improved accuracy of clinical diagnosis, and inclusion of targeted molecular therapies. [Display omitted] DisclosuresKobos:Janssen: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Roshal:BD Biosciences: Consultancy. Zhong:foundation medicine: Employment. Balasubramanian:Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation medicine: Employment. Vergilio:Foundation Medicine: Employment. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Dasatinib Inhibits Actin Fiber Reorganization and Promotes Endothelial Cell Permeability through RhoA-Rock Pathway

BackgroundThe balanced translocation of 5′ segment of the breakpoint cluster region (BCR) gene on chromosome 22 to 3′ segment of the Abelson leukemia virus (ABL) gene on chromosome 9 in chronic myeloid leukemia (CML), results in the formation of the BCR-ABL hybrid fusion gene, which has constitutively upregulated tyrosine kinase activity. Imatinib mesylate, a small molecule developed specifically to inhibit BCR-ABL kinase activity, has revolutionized the treatment of CML. Despite the overwhelming success, about 20% of patients develop resistance to imatinib due to point mutations in the BCR-ABL kinase domain. Dasatinib is one of the second generation tyrosine kinase inhibitors (TKI) that was developed to overcome the resistance to imatinib. However, dasatinib inhibits, in addition to BCR-ABL kinase, other tyrosine kinases such as Src family kinases (SFKs), leading to its unique side effects. Clinically, one of the most common non-hematologic side effects of dasatinib is peripheral edema, pleural effusion and pulmonary hypertension and it is seen 20% of patients treated with dasatinib. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells in vitro and in vivo.MethodsIn vitro endothelial permeability assays were performed with endothelial cells grown on transwell polycarbonate membranes inserts. FITC-Dextran was added to the upper chamber followed by dasatinib (0-100 ng/ml) or vehicle (DMSO) and at various time intervals, the medium in the lower chamber was collected and the fluorescence intensity was measured. In some experiments, the monolayers were incubated with ROCK1 inhibitor y27632 for 10 minutes before the addition of dasatinib. RhoA activity assay was performed and quantified using the RhoA activation assay kit. Spreading of control and dasatinib treated endothelial cells were quantified by Image J software. Actin cytoskeleton was visualized by Immunocytochemistry. Phosphorylation of cytoskeletal proteins were assessed by Western blot with phospho specific antibodies. In vivo endothelial permeability was measured by quantifying the extravasation of intravenously administered Evans Blue to lung parenchyma in mice.ResultsThe permeability of human microvascular endothelial cells (HMEC1) to FITC-dextran increases in Transwell chambers within 5 minutes following the addition of therapeutic concentrations of dasatinib. These changes in permeability are associated with increased activation of RhoA GTPase and its effector Rho associated coiled-coil kinase I (ROCKI). RhoA inhibitor C3 almost completely inhibits dasatinib-induced increase in permeability. Under similar conditions, imatinib had no effect on permeability and it does not activate RhoA. Since integrin-induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impairs endothelial cell spreading in a concentration-dependent manner and induces disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibits tyrosine phosphorylation of p130 Crk associated substrate (p130cas), paxillin and vinculin. These results suggest inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. In vitro, consistent with these findings, dasatinib-induced increase in the permeability is blocked by ROCK inhibitor y-23632. In vivo administration of y-27632 significantly inhibits the dasatinib-induced extravasation of Evans Blue to lung parenchyma in mice. Furthermore, dasatinib-induced microvascular permeability is attenuated in ROCK1-deficient mice.ConclusionsOur findings suggest that dasatinib inhibits actin fiber reorganization and promotes endothelial permeability through RhoA-ROCK pathway and ROCK inhibitors could serve as a therapeutic modality to ameliorate the dasatinib-induced pulmonary changes. DisclosuresNo relevant conflicts of interest to declare.

Abstract 727: Signaling redundancy between EGFR and c-Met: molecular analysis of concurrent inhibition of c-Src and therapeutic potential against prostate cancer

Abstract Overexpression of growth factor receptors is often associated with advanced stage disease and poor prognosis. Importantly, they can interact with other growth factor receptors and non-receptor tyrosine kinases to promote proliferation, survival, invasion and metastasis. One such signaling crosstalk occurs between the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor (c-Met) that are overexpressed in tumours. The amplification of c-Met in tumours overexpressing EGFR leads to signaling redundancy that causes resistance to EGFR inhibitors. Furthermore, the non-receptor tyrosine kinase c-Src has been shown to synergize with EGFR to promote tumour progression and to mediate the crosstalk between EGFR and c-Met. In order to investigate the mechanisms associated with the EGFR-c-Met-c-Src axis, we sought to identify tumour cell lines responding to the dual-inhibition of EGFR and c-Met and the triple-inhibition of EGFR, c-Met and c-Src using pharmacological inhibitions. We identified two androgen independent prostate cancer cell lines with exquisite sensitivity to EGFR-, c-Met-, c-Src-based combinations: DU145 and PC3. The results showed that the combination of crizotinib (c-Met inhibitor) + gefitinib (EGFR inhibitor) was 2-5-fold more potent (DU145 IC50 = 1.9 μM, PC3 IC50 = 1.9 μM) than the drugs alone in growth inhibition assay. Furthermore, the addition of dasatinib (c-Src inhibitor) to the crizotinib + gefitinib combination further enhanced its potency (DU145 IC50 = 0.003 μM, PC3 IC50 = 0.009 μM). When compared with individual drugs, equieffective combinations of gefitinib with crizotinib showed 2-3-fold superior potency in blocking invasion and inducing cytotoxicity. More importantly, the addition of dasatinib to the crizotinib + gefitinib combination significantly enhanced anti-invasive potency and cell-killing by apoptosis. Analysis of signaling crosstalk between the three kinases showed that dasatinib alone or in combination induced re-phosphorylation of c-Src, EGFR, c-Met and STAT3, particularly after 2-6h of drug exposure. The results indicate that these apparent compensatory re-phosphorylation mechanisms induced by dasatinib did not affect the overall potency of the triple combination. Given the role of these three tyrosine kinases in driving proliferation, invasion and survival of cancer cells, our work suggests that triple-inhibition is required for optimal antitumour activity in cells co-expressing EGFR and c-Met. Citation Format: Suman Rao, Anne-Laure Larroque-Lombard, Ben Allal, Bertrand J. Jean-Claude. Signaling redundancy between EGFR and c-Met: molecular analysis of concurrent inhibition of c-Src and therapeutic potential against prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2015-727

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Dasatinib combined with gemcitabine (Gem) in patients (pts) with locally advanced pancreatic adenocarcinoma (PaCa): Design of CA180-375, a placebo-controlled, randomized, double-blind phase II trial.

TPS4134 Background: Dasatinib, a potent oral BCR-ABL and SRC family kinase (SFK) inhibitor, is approved for first- and second-line therapy of Philadelphia chromosome-positive chronic phase chronic myeloid leukemia (CML) in pts with newly diagnosed CML or CML resistant/intolerant to prior therapy. SRC expression and activity is upregulated in PaCa and correlates with reduced survival in resected high-grade PaCa (Morton, Gastroenterology 2010) and resistance to Gem, a PaCa standard of care (Duxbury, J Am Coll Surg 2004). In preclinical PaCa studies, inhibition of SFKs with dasatinib reduces tumor cell proliferation, migration, and invasion; increases apoptosis; sensitizes cells to Gem; and inhibits development of metastases in vivo either alone or in combination with Gem (Duxbury, Clin Cancer Res 2004; Duxbury, J Am Coll Surg 2004; Nagaraj, Mol Cancer Ther 2010; Morton, op cit). Phase I clinical studies of dasatinib and Gem therapy in PaCa have demonstrated feasibility and suggested efficacy of the combination (Uronis, ASCO 2009, abstract e15506). Methods: This double-blind phase II study tests whether addition of dasatinib to Gem is tolerable and improves efficacy in pts with histologically/cytologically confirmed unresectable locally advanced nonmetastatic PaCa. Eligible pts, aged ≥18 years with Eastern Cooperative Oncology Group performance status ≤1 and adequate organ function, are randomized 1:1 to Gem 1000 mg/m2 IV once weekly (Weeks 1–3 of a 4-week cycle) plus either dasatinib 100 mg once daily or matched placebo. Pts are treated until progression, unacceptable toxicity, withdrawal of consent, or study termination. The primary endpoint is OS, and secondary endpoints are progression-free survival and safety. Exploratory endpoints include freedom from distant metastases, measures of pain and fatigue, overall response rate, and carbohydrate antigen 19-9. Final study analysis will be conducted after 135 deaths; all pts will be followed for survival. To date, 23/200 pts have enrolled; estimated primary completion date is March 2013. ClinicalTrials.gov identifier: NCT01395017.

Abstract 2702: Effects of Src and IGF1R inhibition on acquired endocrine therapy resistant breast cancer

Abstract Effective standard therapy for hormone receptor-positive (HR+) breast cancer targets the estrogen receptor (ER). However, a significant proportion of women with HR+ breast cancer relapse. Furthermore, the majority of women with metastatic HR+ breast cancer develop resistance to endocrine therapy. Adaptive cross-talk between ER and growth factor receptor pathways including IGF1R and Src is involved in acquired endocrine therapy resistance. To determine effects of adding dasatinib (D), Src inhibitor, and/or anti-IGF1R Ab MK0646 (M) to the ER antagonist fulvestrant (F) in an acquired endocrine therapy resistant breast cancer, we used long-term estrogen-deprived (LTED) derivative of ER+ breast cancer cell lines, MCF-7/LTED and HCC-1428/LTED. Parental and LTED cells were treated with variable combinations of F, D and M, followed by determinations of drug effects on cell growth, migration, and invasion in vitro and tumor growth in vivo. We demonstrated that LTED cells were resistant to F alone, comparing parental cells (P&amp;lt;0.05) in cell growth, migration and invasion. Addition of D to F appeared to be synergistic in all systems including cell growth, cell migration and invasion. However, the addition of M to F or to F+D appeared inhibition on cell growth in monolayer with low-serum medium, but not to significantly add inhibitory effects on cell growth in 3D and cell migration and invasion. The effects of IGF-1R antibody, MK0646, on cells growth in monolayer showed not consistent with 3D, which might be due to growth factors in the matrigel, although we used growth factors decreased matrigel. To verify the effects of the drugs on tumor growth in vivo, we developed xenograft models using MCF-7/LTED cells and treated the mice with F, D, and M along or variable combinations. The study demonstrated that all the combinations of F+D+M, F+D, and F+M (from P&amp;lt; 0.05 to &amp;lt;0.001 in viable time points) show inhibited the tumor growth comparing vehicle or F alone. The inhibitory effect of F+D+M is higher than F+D and F+M. In summary, the addition of dasatinib to fulvestrant appeared to be synergistic in all systems including cell growth, cell migration and invasion and tumor growth in vivo. The addition of MK0646 to fulvestrant mildly inhibited MCF-7/LTED cell growth and xenografts growth, but not cell migration and cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2702. doi:1538-7445.AM2012-2702

Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

AbstractRecent evidence suggests chronic myeloid leukemia (CML) stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiologic growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%; however, the surviving fraction was enriched for primitive leukemic cells capable of growth in a long-term culture-initiating cell assay and expansion on removal of dasatinib and addition of growth factors. Together, these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance.