Papers by Steven Kornblau
PubMed, Aug 1, 1998
A prior retrospective study suggested that the level of retinoblastoma protein (RB) expression wa... more A prior retrospective study suggested that the level of retinoblastoma protein (RB) expression was prognostic for survival in acute myelogenous leukemia (AML). Individuals with no/low RB protein expression were considered to have loss of RB function, and those with maximally phosphorylated (maxphos) RB were also felt to have nonfunctional RB. To confirm this, we prospectively investigated whether the level of RB expression was prognostic in AML in a larger cohort of patients. RB level was measured by Western blot and immunohistochemical analysis on peripheral blood samples from 210 newly diagnosed AML patients. Patients were divided into three groups based on the level of RB protein expression (i.e., no or low, elevated, and maxphos) or into two groups on the basis of presumed RB function, altered function (AF-RB, low and maxphos RB), versus normal function (NF-RB, elevated RB). By combined results of Western blot and immunohistochemical analysis, 20%, 65%, and 15% of patients had low, elevated, and maxphos RB, respectively. Most patients with acute promyelocytic leukemia (APL) with a French-American-British classification of M3 were in the low RB group, likely reflecting a lower proliferative rate of promyelocytes. Analysis was performed with and without these APL patients. The median survival was significantly shorter for both patients with low RB expression (48 weeks, P = 0.05, including APL patients; 34 weeks, corrected P = 0.008, with APL patients excluded) and maxphos RB expression (51 weeks, P = 0.007) compared to those with elevated RB expression (122 weeks including and 98 weeks excluding APL patients). Differences were greatest among patients with nonfavorable prognosis cytogenetics (median survival, 34 weeks versus 85 weeks; corrected P = 0.001 for AF-RB versus NF-RB). Remission duration was also significantly shorter for non-APL patients with AF-RB versus NF-RB (median survival, 36 weeks versus not reached; corrected P = 0.02). In multivariate analyses, including cytogenetics, performance status, age, antecedent hematological disorder, and RB status, with and without APL patients included, no/low and maxphos-RB protein expression were independent predictors for poorer survival. This prospective study confirms that the level of expression of RB is a strong prognostic factor in AML, with an inferior survival experience being associated with no/low RB and maxphos RB expression. Therefore, therapeutic decisions based on the level of RB expression may be indicated, and protocols to incorporate this are currently under development.
Leukemia & Lymphoma, Oct 13, 2016
ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian h... more ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian histone H3K4 methyltransferase complexes, including Mixed Lineage Leukemia (MLL) complexes. ASH2L protein levels in primary leukemia patient samples have not yet been defined. We analyzed ASH2L protein expression in 511 primary AML patient samples using reverse phase protein array technology. We discovered that ASH2L expression is significantly increased in a subset of patients carrying FLT3 mutations. Furthermore, we observed that low levels of ASH2L are associated with increased overall survival. We also compared ASH2L levels to the expression of 230 proteins previously analyzed on this array. ASH2L expression was inversely correlated with 32 proteins, mostly involved in cell adhesion and cell cycle inhibition, while a positive correlation was observed for 50 proteins, many of which promote cell proliferation. Together, these results indicate that a lower level of ASH2L protein is beneficial to AML patients.
Outcomes of adults with AML remain unsatisfactory. To explore the role of the AKT signaling pathw... more Outcomes of adults with AML remain unsatisfactory. To explore the role of the AKT signaling pathway in AML, we examined samples from AML patients (pts) utilizing reverse phase protein arrays (RPPAs). High levels of the phosphorylated Ser473 form of AKT were associated with shorter remission (p=0.03) and shorter overall survival (p=0.09) in 92 newly diagnosed AML pats with intermediate-risk cytogenetics undergoing induction chemotherapy, identifying AKT as rational drug target. We studied the effects of MK-2206, an investigative oral, highly specific allosteric inhibitor of the 3 isoforms of human AKT, in human AML. MK-2206 dephosphorylated AKT and inhibited growth of AML cell lines and primary AML blasts at low-micromolar concentrations. We then conducted a phase 2 trial of MK-2206 (200 mg once weekly) in adult pats who had failed 1 other therapy for persistent AML or relapsed after a prior CR <12 months, a population with expected CR rates of <5%. Nineteen pats (18 evaluable) with median age 70 (31-86) years were enrolled, one of whom had isolated extramedullary relapse. Nine pats had prior MDS; 5 had normal cytogenetics, 5 had complex cytogenetics or del7, 3 trisomy 8, and 6 had other cytogenetic changes; no patients had FLT3 mutations. Median number of cycles was 2; 4 pats completed ≥ 3 cycles. One response (a CRp) was observed (95% CI: 0-17%). This pat was a 77-yo man with a normal karyotype AML and prior history of CMML, who failed 2 prior regimens including high-dose cytarabine, and continued on MK-2206 for a total of 11 cycles after which he progressed. The most common grade 3/4 adverse event (AE) related to MK-2206 was pruritic rash occurring in 6/18 pats (30%). Other frequent MK-2206-related AEs were rash (n=6; grade 1/2), hyperglycemia (n=12; all grade 1/2 except for one grade 4), QTC prolongation (n=3; all grade 1). We used RPPAs to assess target inhibition in peripheral blood (PB) in 8 pats prior to and 24 hours after the first MK-2206 dose, and in bone marrow (BM) prior to and after 28 days in 5 pats. We examined (a) the direct target, (b) immediate downstream targets (20 total), and (c) all 157 proteins assayed. Considered alone, the decrease in Ser473 AKT was significant (t-test p=0.018). The larger sets show no significant changes after adjusting for multiple testing. Decrease in Ser473 AKT (median 28%; range, 12-45%) was seen in 5/8 PB and 3/5 BM samples; and in Thr308 pAKT in 5/8 PB and 4/5 BM samples. In turn, changes in AKT targets were less prevalent (downregulation of pFOX3A in 3/5, pPRAS40 in 2/5, pS6K in 4/5 and p4EBP1 in 2/5 PB samples), possibly due to incomplete AKT inhibition. Our results indicate single-agent MK-2206 has limited activity in this pat population, with rash being the dose-limiting toxicity precluding further dose escalation. The modest inhibitory effects on AKT and its downstream targets in our RPPA studies suggest that other approaches to block this pathway should be explored in the AML setting. Citation Format: Marina Y. Konopleva, Roland B. Walter, Stefan Faderl, Elias Jabbour, Zhihong Zeng, Gautam Borthakur, Peter Ruvolo, Xuelin Huang, Tapan Kadia, Jennie Feliu, Jan A. Burger, Michael Andreeff, Wenbin Liu, Steven M. Kornblau, Keith Baggerly, Elihu Estey, Hagop Kantarjian. Phase II study of the oral AKT inhibitor, MK-2206, for acute myeloid leukemia (AML) in second relapse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2013-LB-293
Haematologica, Jun 28, 2017
T he bone marrow microenvironment is known to provide a survival advantage to residual acute myel... more T he bone marrow microenvironment is known to provide a survival advantage to residual acute myeloid leukemia cells, possibly contributing to disease recurrence. The mechanisms by which stroma in the microenvironment regulates leukemia survival remain largely unknown. Using reverse-phase protein array technology, we profiled 53 key protein molecules in 11 signaling pathways in 20 primary acute myeloid leukemia samples and two cell lines, aiming to understand stroma-mediated signaling modulation in response to the targeted agents temsirolimus (MTOR), ABT737 (BCL2/BCL-XL), and Nutlin-3a (MDM2), and to identify the effective combination therapy targeting acute myeloid leukemia in the context of the leukemia microenvironment. Stroma reprogrammed signaling networks and modified the sensitivity of acute myeloid leukemia samples to all three targeted inhibitors. Stroma activated AKT at Ser473 in the majority of samples treated with single-agent ABT737 or Nutlin-3a. This survival mechanism was partially abrogated by concomitant treatment with temsirolimus plus ABT737 or Nutlin-3a. Mapping the signaling networks revealed that combinations of two inhibitors increased the number of affected proteins in the targeted pathways and in multiple parallel signaling, translating into facilitated cell death. These results demonstrated that a mechanism-based selection of combined inhibitors can be used to guide clinical drug selection and tailor treatment regimens to eliminate microenvironment-mediated resistance in acute myeloid leukemia.
Clinical Cancer Research, Apr 15, 2014
Purpose: Recent studies suggested that AKT activation might confer poor prognosis in acute myelog... more Purpose: Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore, explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206. Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA). Results: In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in AML cell lines and primary AML blasts. We then treated 19 patients with MK-2206 but, among 18 evaluable participants, observed only 1 (95% confidence interval, 0%-17%) response (complete remission with incomplete platelet count recovery), leading to early study termination. The most common grade 3/4 drugrelated toxicity was a pruritic rash in 6 of 18 patients. Nevertheless, despite the use of MK-2206 at maximum tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12%-45%) and limited inhibition of downstream targets. Conclusions: Although preclinical activity of MK-2206 can be demonstrated, this inhibitor has insufficient clinical antileukemia activity when given alone at tolerated doses, and alternative approaches to block AKT signaling should be explored. Clin Cancer Res; 20(8); 2226-35. Ă“2014 AACR.
Blood, Nov 15, 2013
Introduction Bruton′s tyrosine kinase (BTK) is a member of TEC family of non-receptor tyrosine ki... more Introduction Bruton′s tyrosine kinase (BTK) is a member of TEC family of non-receptor tyrosine kinases. BTK is mostly expressed in hematopoietic cell lineages, except in T cells. It plays a particularly important role in B cell development and is present at almost all stages of their maturation, disappearing only in plasma cells. BTK is an essential kinase downstream of pre-B cell (pre-BCR) and B cell receptors (BCR) promoting proliferation, differentiation and survival of B cells. Methods Surface protein expression in B-cell acute lymphoblastic leukemia (B-ALL) cell lines was assessed by flow cytometry using PE conjugated anti-CD179a, anti-CD179b (BioLegend) and anti-CD22 (BD Pharmingen). To investigate effects of the BTK inhibitor ibrutinib (PCI-32765) on constitutive pre-BCR signaling RCH-ACV cells were pretreated with 0.1% DMSO or increasing concentrations of the drug (0.0001, 0.001, 0.01, 0.1, 1.0 μM) for 1 hour and lysed in RIPA buffer. To induce pre-BCR signaling in pretreated cells they were incubated with 10 μg/ml of anti-Igμ for 30 minutes. Intracellular calcium mobilization was measured by using the fluorogenic probe Fluo3-AM (Invitrogen). RCH-ACV cells pretreated with 1 μM ibrutinib for 72 hours were subjected to gene expression profile analysis on HT-12 v4 Expression BeadChip (Illumina). Results Previously we explored the effects of ibrutinib in B-ALL cell lines and primary samples. Ibrutinib induced only low levels of apoptosis in B-ALL cell lines, but significantly inhibited their proliferation. RCH-ACV and SMS-SB were the most sensitive cell lines with half maximal inhibitory concentrations of ibrutinib of 0.6 and 0.4 μM found in XTT cell proliferation assay. Interestingly, both cell lines expressed a pre-B cell immunophenotype with pre-BCR surface expression. Next, we explored the effect of BTK inhibition on constitutive and induced pre-BCR signaling. Treatment of RCH-ACV cells with varying concentrations of ibrutinib resulted in decreased levels of pBTK, pAKT, pS6 and pSYK. The lowest concentration of ibrutinib needed to observe complete disappearance of pBTK (Y223) and any reduction of other phospho-proteins was 10 nM, however the maximum effect was achieved with 1 μM ibrutinib. Upon pre-BCR crosslinking with anti-Igμ elevated levels of pSYK, pBTK, pAKT, pS6 and pERK were detected in RCH-ACV. Pretreatment of the cells with ibrutinib greatly reduced this effect. As calcium mobilization is another important indicator of B cell activation upon pre-BCR stimulation, we evaluated ibrutinib in calcium flux assays. Pretreatment with 1 μM ibrutinib effectively abrogated anti-Igμ induced calcium flux in pre-B ALL cell lines. Gene expression profile analysis of RCH-ACV cells after 72 hours of incubation with 1 μM ibrutinib showed down-regulation of pre-BCR related genes such as PTPN6 (SHP-1), Bcl6 and CD22. Flow cytometry analysis confirmed the down-regulation of the inhibitory co-receptor CD22 in pre-B ALL cell lines after incubation with ibrutinib. The down-regulation of SHP-1 protein was verified by western blotting. Conclusions The results indicate that ibrutinib reduces the pre-B ALL cell proliferation by inhibiting constitutive and/or induced pre-BCR signaling. Observed down-regulation of CD22 and SHP-1, known negative regulators of BCR signaling, suggests a possible mechanism of cell adaptation to the presence of the BTK inhibitor. Taken together, these data provide a rationale for clinical testing of ibrutinib in B-ALL with active pre-BCR signaling. Disclosures: O'Brien: Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment. Burger:Pharmacyclics: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.
Blood, 2013
Introduction Bruton′s tyrosine kinase (BTK) is a member of TEC family of non-receptor tyrosine ki... more Introduction Bruton′s tyrosine kinase (BTK) is a member of TEC family of non-receptor tyrosine kinases. BTK is mostly expressed in hematopoietic cell lineages, except in T cells. It plays a particularly important role in B cell development and is present at almost all stages of their maturation, disappearing only in plasma cells. BTK is an essential kinase downstream of pre-B cell (pre-BCR) and B cell receptors (BCR) promoting proliferation, differentiation and survival of B cells. Methods Surface protein expression in B-cell acute lymphoblastic leukemia (B-ALL) cell lines was assessed by flow cytometry using PE conjugated anti-CD179a, anti-CD179b (BioLegend) and anti-CD22 (BD Pharmingen). To investigate effects of the BTK inhibitor ibrutinib (PCI-32765) on constitutive pre-BCR signaling RCH-ACV cells were pretreated with 0.1% DMSO or increasing concentrations of the drug (0.0001, 0.001, 0.01, 0.1, 1.0 μM) for 1 hour and lysed in RIPA buffer. To induce pre-BCR signaling in pretreate...
Proteomics, 2018
Posttranslational histone tail modifications are known to play a role in leukemogenesis and are t... more Posttranslational histone tail modifications are known to play a role in leukemogenesis and are therapeutic targets. A global analysis of the level and patterns of expression of multiple histone-modifying proteins (HMP) in acute myeloid leukemia (AML) and the effect of different patterns of expression on outcome and prognosis has not been investigated in AML patients. Here we analyzed 20 HMP by reverse phase protein array (RPPA) in a cohort of 205 newly diagnosed AML patients. Protein levels were correlated with patient and disease characteristics, including survival and mutational state. We identified different protein clusters characterized by higher (more on) or lower (more off) expression of HMP, relative to normal CD34+ cells. On state of HMP was associated with poorer outcome compared to normal-like and a more off state. FLT3 mutated AML patients were significantly overrepresented in the more on state. DNA methylation related mutations showed no correlation with the different ...
Molecular cancer research : MCR, Jan 18, 2018
Heterogeneity in the genetic landscape of pediatric acute myeloid leukemia (AML) makes personaliz... more Heterogeneity in the genetic landscape of pediatric acute myeloid leukemia (AML) makes personalized medicine challenging. As genetic events are mediated by the expression and function of proteins, recognition of recurrent protein patterns could enable classification of pediatric AML patients and could reveal crucial protein dependencies. This could help to rationally select combinations of therapeutic targets. To determine if protein expression levels could be clustered into functionally relevant groups, custom Reverse Phase Protein Arrays (RPPA) were performed on pediatric AML (n=95) and CD34+ normal bone marrow (n=10) clinical specimens using 194 validated antibodies. To analyze proteins in the context of other proteins, all proteins were assembled into 31 Protein Functional Groups (PFG). For each PFG an optimal number of protein clusters was defined that represented distinct transition states. Block clustering analysis revealed strong correlations between various protein clusters...
Haematologica, Jan 15, 2018
Mesenchymal stromal cells support acute myeloid leukemia cell survival in the bone marrow microen... more Mesenchymal stromal cells support acute myeloid leukemia cell survival in the bone marrow microenvironment. Protein expression profiles of acute myeloid leukemia-derived mesenchymal stromal cells are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from acute myeloid leukemia mesenchymal stromal cells (n = 106) with mesenchymal stromal cells from healthy donors (n = 71). Protein expression differed significantly between the two groups with nineteen proteins overexpressed in leukemia stromal cells and nine overexpressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these twenty-eight proteins revealed three protein constellations whose variation in expression defined four mesenchymal stromal cells protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cells populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and rem...
Haematologica, Jan 28, 2017
The bone marrow microenvironment is known to provide a survival advantage to residual acute myelo... more The bone marrow microenvironment is known to provide a survival advantage to residual acute myeloid leukemia cells, possibly contributing to disease recurrence. The mechanisms by which stroma in the microenvironment regulates leukemia survival remain largely unknown. Using reverse phase-protein array technology, we profiled 53 key protein molecules in 11 signaling pathways in 20 primary acute myeloid leukemia samples and two cell lines, aiming to understand stroma-mediated signaling modulation in response to the targeted agents temsirolimus (MTOR), ABT737 (BCL2/BCL-XL), and Nutlin-3a (MDM2), and to identify the effective combination therapy targeting acute myeloid leukemia in the context of the leukemia microenvironment. Stroma reprogrammed signaling networks and modified the sensitivity of acute myeloid leukemia samples to all three targeted inhibitors. Stroma activated AKT at Ser473 in the majority of samples treated with single-agent ABT737 or Nutlin-3a. This survival mechanism w...
Leukemia & Lymphoma, 2016
ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian h... more ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian histone H3K4 methyltransferase complexes, including Mixed Lineage Leukemia (MLL) complexes. ASH2L protein levels in primary leukemia patient samples have not yet been defined. We analyzed ASH2L protein expression in 511 primary AML patient samples using reverse phase protein array technology. We discovered that ASH2L expression is significantly increased in a subset of patients carrying FLT3 mutations. Furthermore, we observed that low levels of ASH2L are associated with increased overall survival. We also compared ASH2L levels to the expression of 230 proteins previously analyzed on this array. ASH2L expression was inversely correlated with 32 proteins, mostly involved in cell adhesion and cell cycle inhibition, while a positive correlation was observed for 50 proteins, many of which promote cell proliferation. Together, these results indicate that a lower level of ASH2L protein is beneficial to AML patients.
Oncotarget, 2016
mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The a... more mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orallyadministered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.
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Papers by Steven Kornblau