Elie Traer

Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

Crosstalk between KIT and FGFR3 Promotes Gastrointestinal Stromal Tumor Cell Growth and Drug Resistance

Kinase inhibitors such as imatinib have dramatically improved outcomes for patients with gastrointestinal stromal tumor (GIST), but many patients develop resistance to these treatments. Although in some patients this event corresponds with mutations in the GIST driver oncogenic kinase KIT, other patients develop resistance without KIT mutations. In this study, we address this patient subset in reporting a functional dependence of GIST on the FGF receptor FGFR3 and its crosstalk with KIT in GIST cells. Addition of the FGFR3 ligand FGF2 to GIST cells restored KIT phosphorylation during imatinib treatment, allowing sensitive cells to proliferate in the presence of the drug. FGF2 expression was increased in imatinib-resistant GIST cells, the growth of which was blocked by RNAi-mediated silencing of FGFR3. Moreover, combining KIT and FGFR3 inhibitors synergized to block the growth of imatinib-resistant cells. Signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to imatinib. Clinically, an IHC analysis of tumor specimens from imatinib-resistant GIST patients revealed a relative increase in FGF2 levels, with a trend toward increased expression in imatinib-naïve samples consistent with possible involvement in drug resistance. Our findings provide a mechanistic rationale to evaluate existing FGFR inhibitors and multikinase inhibitors that target FGFR3 as promising strategies to improve treatment of patients with GIST with de novo or acquired resistance to imatinib.

Ponatinib overcomes FGF2-mediated resistance in CML patients without kinase domain mutations

Development of resistance to kinase inhibitors remains a clinical challenge. Kinase domain mutations are a common mechanism of resistance in chronic myeloid leukemia (CML), yet the mechanism of resistance in the absence of mutations remains unclear. We tested proteins from the bone marrow microenvironment and found that FGF2 promotes resistance to imatinib in vitro. Fibroblast growth factor 2 (FGF2) was uniquely capable of promoting growth in both short- and long-term assays through the FGF receptor 3/RAS/c-RAF/mitogen-activated protein kinase pathway. Resistance could be overcome with ponatinib, a multikinase inhibitor that targets BCR-ABL and FGF receptor. Clinically, we identified CML patients without kinase domain mutations who were resistant to multiple ABL kinase inhibitors and responded to ponatinib treatment. In comparison to CML patients with kinase domain mutations, these patients had increased FGF2 in their bone marrow when analyzed by immunohistochemistry. Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGF receptor inhibition. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance.

Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML

Background Mutations in the gene encoding isocitrate dehydrogenase 1 (IDH1) occur in 6 to 10% of patients with acute myeloid leukemia (AML). Ivosidenib (AG‐120) is an oral, targeted, small‐molecule inhibitor of mutant IDH1. Methods We conducted a phase 1 dose‐escalation and dose‐expansion study of ivosidenib monotherapy in IDH1‐mutated AML. Safety and efficacy were assessed in all treated patients. The primary efficacy population included patients with relapsed or refractory AML receiving 500 mg of ivosidenib daily with at least 6 months of follow‐up. Results Overall, 258 patients received ivosidenib and had safety outcomes assessed. Among patients with relapsed or refractory AML (179 patients), treatment‐related adverse events of grade 3 or higher that occurred in at least 3 patients were prolongation of the QT interval (in 7.8% of the patients), the IDH differentiation syndrome (in 3.9%), anemia (in 2.2%), thrombocytopenia or a decrease in the platelet count (in 3.4%), and leukocytosis (in 1.7%). In the primary efficacy population (125 patients), the rate of complete remission or complete remission with partial hematologic recovery was 30.4% (95% confidence interval [CI], 22.5 to 39.3), the rate of complete remission was 21.6% (95% CI, 14.7 to 29.8), and the overall response rate was 41.6% (95% CI, 32.9 to 50.8). The median durations of these responses were 8.2 months (95% CI, 5.5 to 12.0), 9.3 months (95% CI, 5.6 to 18.3), and 6.5 months (95% CI, 4.6 to 9.3), respectively. Transfusion independence was attained in 29 of 84 patients (35%), and patients who had a response had fewer infections and febrile neutropenia episodes than those who did not have a response. Among 34 patients who had a complete remission or complete remission with partial hematologic recovery, 7 (21%) had no residual detectable IDH1 mutations on digital polymerase‐chain‐reaction assay. No preexisting co‐occurring single gene mutation predicted clinical response or resistance to treatment. Conclusions In patients with advanced IDH1‐mutated relapsed or refractory AML, ivosidenib at a dose of 500 mg daily was associated with a low frequency of grade 3 or higher treatment‐related adverse events and with transfusion independence, durable remissions, and molecular remissions in some patients with complete remission. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02074839.)

Targeting BCL-2 and ABL/LYN in Philadelphia chromosome-positive acute lymphoblastic leukemia

The combination of venetoclax and dasatinib is synergistically effective in treating Philadelphia chromosome–positive ALL. Showing ALL that resistance is futile Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ALL) carries the BCR-ABL translocation; however, despite the existence of drugs that target this translocation, Ph+ALL remains very difficult to treat, and single-drug treatment is usually ineffective. Leonard et al. showed that venetoclax, an apoptosis-promoting drug, is effective in treating this disease and that dasatinib, an inhibitor of BCR-ABL, blocks a common mechanism of resistance to venetoclax. As a result, these two drugs work together synergistically in vitro and in preclinical models, offering a promising approach toward the rational treatment of this disease. Treatment of Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ALL) remains a challenge. Although the addition of targeted tyrosine kinase inhibitors (TKIs) to standard cytotoxic therapy has greatly improved upfront treatment, treatment-related morbidity and mortality remain high. TKI monotherapy provides only temporary responses and renders patients susceptible to the development of TKI resistance. Thus, identifying agents that could enhance the activity of TKIs is urgently needed. Recently, a selective inhibitor of B cell lymphoma 2 (BCL-2), ABT-199 (venetoclax), has shown impressive activity against hematologic malignancies. We demonstrate that the combination of TKIs with venetoclax is highly synergistic in vitro, decreasing cell viability and inducing apoptosis in Ph+ALL. Furthermore, the multikinase inhibitors dasatinib and ponatinib appear to have the added advantage of inducing Lck/Yes novel tyrosine kinase (LYN)–mediated proapoptotic BCL-2–like protein 11 (BIM) expression and inhibiting up-regulation of antiapoptotic myeloid cell leukemia 1 (MCL-1), thereby potentially overcoming the development of venetoclax resistance. Evaluation of the dasatinib-venetoclax combination for the treatment of primary Ph+ALL patient samples in xenografted immunodeficient mice confirmed the tolerability of this drug combination and demonstrated its superior antileukemic efficacy compared to either agent alone. These data suggest that the combination of dasatinib and venetoclax has the potential to improve the treatment of Ph+ALL and should be further evaluated for patient care.

Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance.

Bone marrow stromal cells (BMSC) in acute myeloid leukaemia (AML) contribute to extrinsic drug resistance, generally attributed to cell–cell contact or secreted cytokines (Jacamo et al, 2014). However, a recent report indicates that stromal protection may also occur via other soluble factors (Yang et al, 2014). Extracellular vesicles, such as exosomes, traffic protein and RNA between cells (Valadi et al, 2007). Stromal exosomes were recently shown to confer a proliferative advantage to multiple myeloma cells via transfer of microRNA (miRNA) (Roccaro et al, 2013). The functional impact of stromal exosomes in AML has not been studied. We therefore hypothesized that AML stroma release exosomes that protect leukaemia cells and traffic a distinct subset of miRNA and cytokines. We isolated stromal cells from the marrow aspirates of 20 patients (AML-BMSC; see Table SI for patient characteristics) and five healthy controls (N-BMSC) according to a previously published protocol (Tyner et al, 2013). The cells were fibroblastic, adherent and expressed CD90, but not CD45 epitopes (Fig 1A). Reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of canonical stromal transcripts in both populations (Fig 1B) (Boxall & Jones, 2012). However, quantitative analysis revealed altered expression in AML-BMSC for CXCL12, KITLG and CXCL1, as well as for genes previously reported in modified stroma in myelodysplastic syndrome (IGFBP4, ANGPTL4) (Fig 1C) (Medyouf et al, 2014). The contribution of stromal-derived exosomes has been established in other malignancies (Roccaro et al, 2013). We isolated vesicles using ultracentrifugation and then used vesicle tracking analysis, electron microscopy and Western blotting to demonstrate a vesicle population conforming in size, morphology and tetraspanin membrane proteins to exosomes (Fig 1D) (Valadi et al, 2007). Fig 1 AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity ... Exosome biogenesis allows for selective incorporation of different RNA species (Valadi et al, 2007). We determined the spectrum of BMSC exosome RNA using a Bioanalyser (Agilent, Santa Clara, CA, USA) and observed a relatively greater abundance of small RNAs compared with the parent cell (Fig 1D). Increased levels of MIR155 and MIR375 can independently identify AML patients at high risk for recurrence, with MIR155 contributing to the pathogenesis of several other haematological malignancies (Marcucci et al, 2013; Wang et al, 2013). Reasoning that stromal cell reprogramming in AML leads to unique exosomal miRNA incorporation, we used quantitative RT-PCR to compare the MIR155 and MIR375 levels in stromal cells from 12 patients (4 normal, 8 AML) and the exosomes released during the culture period. Strikingly, all eight AML-BMSC samples showed a statistically significant fold increase in incorporation of MIR155 and MIR375 in the exosomes relative to their parent cells, while no such increase was observed for N-BMSCs (Fig 1E). The comparative enrichment in AML-BMSC exosomes persisted even after 10 passages in tissue culture devoid of leukaemia cells, suggesting a durable change in stromal cell biology (Fig 1E). We then directly compared miRNA content between exosome isolates, independent of the parent cell content, and found MIR155, but not MIR375, was consistently elevated in AML-BMSC exosomes (Fig 1F). Taken together, the data indicate that exosomes released from AML-BMSCs are selectively enriched for miRNA that signify disease risk status in AML. Cytokines and growth factors contribute to leukaemia niche function (Yang et al, 2014). We reasoned that exosome- associated and directly secreted cytokine alterations from AML-BMSCs exist. In a multiplex screening approach using bead-based technology (Luminex, Austin, TX, USA) and enzyme-linked immuno-sorbent-assay (ELISA) kits (Life Technologies, Carlsbad, CA, USA), we studied the concentration of cytokines in the exosomes and in the vesicle-depleted soluble protein fraction of conditioned media from AML-BMSCs (n = 10) and N-BMSCs (n = 3). Exosomal cytokine concentrations ranged from 1·6 pg/ml (B-FGF) to >10 000 pg/ml [interleukin (IL) 8], while other cytokines were undetected in exosomes or in the soluble protein fraction from BMSCs (IL2, IL5, IL17A, TNF, IFNG). We found statistically significant enrichment of epidermal growth factor (P < 0·05), as well as a relative depletion of hepatocyte growth factor (P = 0·08) in exosomes from AML-BMSC, changes that were not observed in the soluble protein fraction when compared to N-BMSCs (Fig 2A). Concentrations of other cytokines, whether exosome-associated or freely secreted, did not differ between the two populations. We separately analysed TGFB1, given its correlation with treatment response in AML (Hong et al, 2014). We found TGFB1 at concentrations ranging from 200 to 2000 pg/ml in exosomes from 10/10 AML-BMSC samples, but below the level of assay detection in exosomes from N-BMSCs (Fig 2B). Taken together, our results suggest that stromal cells in AML modify their exosome-associated cytokine concentrations in a manner independent from the directly secreted fraction. Fig 2 Exosomes from AML-BMSCs have altered cytokine levels and confer chemo-protection to AML cells: (A) Box-and-Whisker plot of a multiplex protein assay displaying the relative concentration of 19 cytokines in exosomes and in the vesicle-depleted soluble ... Our findings, along with existing reports on general stromal protection in AML, led us to hypothesize that stromal exosomes alter chemo-resistance in AML cells. Representing a standard component of AML therapy, we treated MOLM-14 FLT3 internal tandem duplication (FLT3-ITD+) AML cells with the nucleoside analogue cytarabine after exposure to exosomes from AML-BMSC, N-BMSC or control media (Fig 2C). The data provide the first evidence, to our knowledge, that exosomes from both AML patients and controls (Total n = 10, 6 AML-BMSC, 4 N-BMSC) protect AML cells from cytarabine. Further, when AML cells were treated with the FLT3 inhibitor AC220 after exposure, only AML-BMSC exosomes significantly protected AML cells (n = 6), while N-BMSC exosomes provided no such protective effect (n = 3) (Fig 2C). Our data in aggregate suggest that stromal cells in AML patients undergo modification that includes alterations in function as well as the protein and RNA present in the exosomes they release. We report a first demonstration that stromal cells in AML release exosomes enriched for known clinical risk factors, including TGFB1, MIR155 and MIR375. Finally, our data add stromal exosome trafficking as a candidate mechanism for extrinsic chemo-resistance within the niche in AML, with differential protection against kinase pathway inhibition observed only by AML-BMSC exosomes. Such unique protection could occur, for example, by exosomal miRNA-directed down-regulation of promoters of apoptosis or cell differentiation, thereby releasing the leukaemia cell from kinase pathway dependence. Future studies in this area may uncover new mediators of such resistance.

Molecularly targeted drug combinations demonstrate selective effectiveness for myeloid- and lymphoid-derived hematologic malignancies

Significance Mononuclear cells obtained from freshly isolated patient samples with various hematologic malignancies were evaluated for sensitivities to combinations of drugs that target specific cell-signaling pathways. The diagnostic, genetic/cytogenetic, and cellular features of the patient samples were correlated with effective drug combinations. For myeloid-derived tumors, such as acute myeloid leukemia, several combinations of targeted agents that include a kinase inhibitor and venetoclax, a selective inhibitor of BCL2, are effective. Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.

FGF2 from Marrow Microenvironment Promotes Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia

Potent FLT3 inhibitors, such as quizartinib (AC220), have shown promise in treating acute myeloid leukemia (AML) containing FLT3 internal tandem duplication (ITD) mutations. However, responses are not durable and resistance develops within months. In this study, we outline a two-step model of resistance whereby extrinsic microenvironmental proteins FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) protect FLT3-ITD+ MOLM14 cells from AC220, providing time for subsequent accumulation of ligand-independent resistance mechanisms. FL directly attenuated AC220 inhibition of FLT3, consistent with previous reports. Conversely, FGF2 promoted resistance through activation of FGFR1 and downstream MAPK effectors; these resistant cells responded synergistically to combinatorial inhibition of FGFR1 and FLT3. Removing FL or FGF2 from ligand-dependent resistant cultures transiently restored sensitivity to AC220, but accelerated acquisition of secondary resistance via reactivation of FLT3 and RAS/MAPK signaling. FLT3-ITD AML patients treated with AC220 developed increased FGF2 expression in marrow stromal cells, which peaked prior to overt clinical relapse and detection of resistance mutations. Overall, these results support a strategy of early combination therapy to target early survival signals from the bone marrow microenvironment, in particular FGF2, to improve the depth of response in FLT3-ITD AML. Cancer Res; 76(22); 6471-82. ©2016 AACR.

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70–80% of patients with JAK2‐V617F‐negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C‐terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium‐binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL‐3‐independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2‐V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll‐like receptor agonist. These effects are not dependent on the novel C‐terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2‐V617F‐negative essential thrombocytosis and primary myelofibrosis. Am. J. Hematol. 91:211–219, 2016.

How much and how long: tyrosine kinase inhibitor therapy in chronic myeloid leukemia.

As the first clinically successful tyrosine kinase inhibitor (TKI), imatinib pioneered a new approach to treating patients with cancer. Dramatic results from chronic myeloid leukemia (CML) clinical trials spurred the development of TKIs for other malignancies such as acute myeloid leukemia as well as kidney and lung cancer. In CML, imatinib resistance led to the rapid development of dasatinib and nilotinib, more potent second-generation ABL kinase inhibitors that can often overcome imatinib resistance. While the clinical efficacy of TKIs in CML is well established, a number of important questions remain about the optimal dose and duration of therapy. Even the best initial dose for imatinib is still under investigation. Although laboratory and clinical studies had led to the prevailing view that continual inhibition of the BCR-ABL kinase was required for optimal efficacy, recent data on dasatinib have upended this notion and have led to a change in the recommended dosing schedule. The availability of dasatinib and nilotinib also begs the question of whether they might be superior to imatinib as first-line agents. Finally, the question of whether it may be possible to stop TKI therapy at least in some patients with CML has attracted considerable attention. More than 10 years after the introduction of imatinib, optimization of TKI therapy for CML continues.