A phase I/II trial of Erlotinib in higher risk myelodysplastic syndromes and acute myeloid leukemia after azacitidine failure.

Survival after azacitidine (AZA) failure in higher-risk myelodysplastic syndromes (MDS) is poor and new treatment options are needed. Erlotinib, an oral inhibitor of the epidermal-growth-factor-receptor (EGFR), has shown in preclinical models some efficacy in higher risk MDS and acute myeloid leukemia (AML). In this phase I/II trial, 30 patients received 100mg/day (n=5) or 150mg/day (n=25) of Erlotinib orally after primary or secondary resistance to AZA treatment. Eighteen MDS and 12 AML patients were treated. This outpatient treatment was well tolerated with limited grade III-IV extra hematological toxicities (skin (n=1), and diarrhea (n=3). Response was observed in 6 patients (20%) including 1 complete remission (CR), 1 marrow CR and 4 hematological improvement (2 erythroid and 2 on platelets). Median duration of response was 5 months. Erlotinib appears to induce a significant number of responses in higher risk MDS/AML having failed AZA treatment. Given the good safety profile of Erlotinib, its combination with other drugs could be tested in the future in MDS and AML.

Erlotinib antagonizes constitutive activation of SRC family kinases and mTOR in acute myeloid leukemia.

Tyrosine kinases such as SRC family kinases (SFKs) as well as the mammalian target of rapamycin (mTOR) serine/threonine kinase are often constitutively activated in acute myeloid leukemia (AML) and hence constitute potential therapeutic targets. Here we demonstrate that the epidermal growth factor receptor (EGFR) inhibitor erlotinib, which has previously been shown to mediate antiproliferative/cytotoxic off-target effects in myelodysplastic syndrome (MDS) and AML blasts, reduces SFK overactivation. Erlotinib induced an arrest in the G 1 phase of the cell cycle that, in cells with constitutive SFK activation, could be recapitulated by chemical inhibition of SFKs with 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-α]pyrimidin-4-amine (PP2). Moreover, erlotinib inhibited the phosphorylation of mTOR targets like p70 (SK6) , stimulated the maturation of the autophagic marker LC3 and promoted the formation of autophagosomes. Notably, PP2 and the mTOR inhibitor rapamycin had a similar cell cycle-arresting activity to erlotinib, but neither of these compounds alone induced significant levels of cell death. Altogether, these results suggest that the therapeutic off-target effect of erlotinib may be linked to, yet cannot be entirely explained by, the inhibition of oncogenic signaling via SFKs and mTOR. Thus, combination therapies with erlotinib and rapamycin might be beneficial for MDS and AML patients.

Tyrosine kinase inhibitors for the treatment of acute myeloid leukemia: delineation of anti-leukemic mechanisms of action.

Initially, tyrosine kinase inhibitors (TKIs) were developed as targeted therapies that would solely interfere with aberrant tyrosine kinase activation in malignant cells. Nevertheless, preclinical and clinical studies demonstrated that TKI also exhibit "off-target" effects, that is effects not mediated by the assumed mechanisms of action. We and others showed that the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib exert potent antineoplastic effects on EGFR-negative myeloblasts from patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Here, we undertook a side-by-side comparison of the anti-leukemic efficacy of four different TKI in MDS and AML. Besides the EGFR inhibitor erlotinib, which served as a point of reference, we employed the dual EGFR/HER2 TKI lapatinib, as well as the multikinase inhibitors dasatinib and sorafenib. All four drugs had anti-leukemic effects on cell line models of MDS/AML in vitro as well as on malignant blasts from MDS/AML patients ex vivo. We explored the biological phenomena underlying this anti-leukemic efficacy. Since it is established that a therapeutic benefit in MDS/AML can be conveyed by induction of cell cycle arrest, apoptosis and/or differentiation, we deciphered the individual contribution of these three phenomena to the anti-leukemic action of each of the four TKI. The concomitant assessment of the panel of TKI enables us thus to define (and quantify) their differential capacity to impact on the three biological phenomena, and provide further evidence that these mechanisms are not solely explained by on-target effects.

Cytotoxic effects of the trifunctional bispecific antibody FBTA05 in ex-vivo cells of chronic lymphocytic leukaemia depend on immune-mediated mechanism.

Monoclonal antibodies such as rituximab and alemtuzumab show considerable therapeutic efficacy in chronic lymphocytic leukaemia (CLL). Aiming to further improve antineoplastic efficacy, the trifunctional bispecific antibody FBTA05 was developed. FBTA05 is thought to function by simultaneously binding B cells and T cells by its variable regions and by recruiting FcγR-positive accessory immune cells by its intact Fc region. As it was previously shown that this antibody shows considerable cytotoxicity towards a spectrum of B-cell lymphoma cell lines, we here tested its potential efficacy ex vivo against malignant B-CLL cells. Therefore, we assessed the capacity of increasing concentrations of FBTA05 to bind to neoplastic cells, to induce cytotoxicity (comparing it with rituximab and alemtuzumab) and cytokine release. We evaluated the results with respect to the extent of CD20 expression, the effector:target cell ratio as well as with the patients' overall effector cell status. Thus, we show that, although FBTA05-elicited cytotoxicity was comparable with that induced by alemtuzumab, it considerably exceeded the antineoplastic effects of rituximab. Noteworthy, FBTA05 shows effective elimination of malignant B cells even if CD20 surface expression is low. Importantly, a high grade of cytotoxicity was associated with the induction of T-cell proliferation and the concomittant release of interferon-γ and interleukin-6, thus overcoming the detrimental effects of an unfavourable effector:target cell ratio. In conclusion, we here present novel evidence for the therapeutic efficacy of the trifunctional, bispecific antibody FBTA05 in CLL and provide evidence for the importance of immune-mediated mechanisms conveying the cytotoxic effects against malignant B lymphocytes.

Hypomethylating agents reactivate FOXO3A in acute myeloid leukemia.

The deregulation of the DNA damage response (DDR) can contribute to leukemogenesis and favor the progression from myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Since hypomethylating agent, notably azacitidine, constitute an efficient therapy for patients with high-risk MDS, we assessed whether such compounds can activate the DDR in malignant blasts. While azacitidine and decitabine had moderate effects on apoptosis and cell cycle progression, both agents induced profound changes in the expression and functionality of DDR-related proteins. Decitabine, and to a lesser degree azacitidine, induced the activation of checkpoint kinases Chk-1 and Chk-2 and the phosphorylation of the DDR-sensor H2AX. In addition, hypomethylating agents were found to cause the dephosphorylation of the transcriptional regulator forkhead box O3, best known as FOXO3A, whose phosphorylation has been related to poor prognosis in AML. The dephoasphorylation of FOXO3A induced by azacitidine or decitabine in malignant blasts was accompanied by the translocation of FOXO3A from the cytoplasm to the nucleus. Upon stimulation with azacitidine, MDS/AML-derived, azacitidine-sensitive SKM-1S cells upregulated FOXO3A and the pro-apoptotic FOXO3A targets BIM and PUMA, and this effect was attenuated or abolished in azacitidine-resistant SMK-1R cells. Altogether, our results suggest that the reactivation of FOXO3A may contribute to the effects of hypomethylating agents in malignant blasts.

Prognostic factors for response and overall survival in 282 patients with higher-risk myelodysplastic syndromes treated with azacitidine.

Prognostic factors for response and survival in higher-risk myelodysplastic syndrome patients treated with azacitidine (AZA) remain largely unknown. Two hundred eighty-two consecutive high or intermediate-2 risk myelodysplastic syndrome patients received AZA in a compassionate, patient-named program. Diagnosis was RA/RARS/RCMD in 4%, RAEB-1 in 20%, RAEB-2 in 54%, and RAEB-t (AML with 21%-30% marrow blasts) in 22%. Cytogenetic risk was good in 31%, intermediate in 17%, and poor in 47%. Patients received AZA for a median of 6 cycles (1-52). Previous low-dose cytosine arabinoside treatment (P = .009), bone marrow blasts > 15% (P = .004), and abnormal karyotype (P = .03) independently predicted lower response rates. Complex karyotype predicted shorter responses (P = .0003). Performance status ≥ 2, intermediate- and poor-risk cytogenetics, presence of circulating blasts, and red blood cell transfusion dependency ≥ 4 units/8 weeks (all P < 10(-4)) independently predicted poorer overall survival (OS). A prognostic score based on those factors discriminated 3 risk groups with median OS not reached, 15.0 and 6.1 months, respectively (P < 10(-4)). This prognostic score was validated in an independent set of patients receiving AZA in the AZA-001 trial (P = .003). Achievement of hematological improvement in patients who did not obtain complete or partial remission was associated with improved OS (P < 10(-4)). In conclusion, routine tests can identify subgroups of patients with distinct prognosis with AZA treatment.

Efficacy and safety of lenalidomide in intermediate-2 or high-risk myelodysplastic syndromes with 5q deletion: results of a phase 2 study.

Higher-risk MDS with del5q carry a poor prognosis. In this phase 2 trial, 47 patients with higher-risk MDS received lenalidomide 10 mg/day. International Prognostic Scoring System was high in 60%, intermediate-2 risk in 40%. del 5q was isolated, with one additional and more than one additional abnormality in 19%, 23%, and 58% patients, respectively. Thirteen (27%) patients achieved hematologic response, including 7 hematologic complete remission (CR) (with complete [4] or partial [3] cytogenetic response), 2 marrow CR and 4 hematologic improvement erythroid, and 12 became red blood cell (RBC) transfusion independent, for a median duration of 6.5 months. Median CR duration was 11.5 months. Six of 9 (67%) patients with isolated del 5q achieved CR, versus 1 of 11 and none of 27 patients with one or more than one additional abnormality, respectively (P < .001). Seven of 20 (35%) with initial platelets more than 100,000/mm(3) obtained CR, compared with none of the 27 with lower platelet counts less than 100,000/mm(3) (P = .001). Our data support a potential role of lenalidomide in higher-risk MDS with isolated del 5q.

Flt3 receptor inhibition reduces constitutive NFkappaB activation in high-risk myelodysplastic syndrome and acute myeloid leukemia.

High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the activation of the anti-apoptotic transcription factor NFkappaB, via the IKK complex. Here, we show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation. Chemical inhibition or knockdown of Flt3 with small interfering RNAs reduced NFkappaB activation in MDS and AML cell lines, as well as in primary CD34(+) bone marrow cells from patients, causing apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK suggested that both kinases act in the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membrane-tethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. Flt3 phosphorylates IKK2 in vitro, and Flt3 inhibition reduced the phosphorylation of IKK2 in MDS or AML cell lines. IKK2 and Flt3 physically associated in MDS and AML cells, and Flt3 inhibition disrupted this interaction. Flt3 inhibition only killed CD34(+) bone marrow cells from high-risk MDS and AML patients, in correlation with blast numbers and NFkappaB activity, yet had no lethal effect on healthy CD34(+) cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of NFkappaB.

A novel effect of DNA methyltransferase and histone deacetylase inhibitors: NFkappaB inhibition in malignant myeloblasts.

Malignant myeloblasts arising in high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the constitutive activation of the anti-apoptotic transcription factor NFkappaB. We found that DNA methyltransferase (DNMT) inhibitors (such as azacytidine and 5-aza-2'-deoxycytidine) and histone deacetylase (HDAC) inhibitors (such as trichostatin and valproic acid) efficiently induced apoptosis in the P39 MDS/AML cell line, correlating with an inhibition of NFkappaB (which translocated from the nucleus to the cytoplasm). This effect was obtained rapidly, within a few hours, suggesting that it was not due to epigenetic reprogramming. Indeed, DNMT and HDAC inhibitors reduced the phosphorylation of the NFkappaB-activating kinase IKKalpha/beta, and this effect was also observed in enucleated cells. Finally, circulating myeloblasts from AML patients treated with the DNMT inhibitor 5-aza-2'-deoxycytidine manifested a rapid (2 hours post-treatment) inhibition of NFkappaB and IKKalpha/beta. Altogether, these results indicate that DNMT and HDAC inhibitors can inhibit the constitutive activation of NFkappaB in malignant myeloblasts in vitro and in vivo through a novel mechanism.

Erlotinib and gefitinib for the treatment of myelodysplastic syndrome and acute myeloid leukemia: a preclinical comparison.

Erlotinib and gefitinib, two inhibitors of the epidermal growth factor receptor (EGFR), can stimulate apoptosis and differentiation of myeloid cell lines that lack EGFR, unveiling a novel, therapeutically exploitable off-target effect of tyrosine kinase inhibitors. Here, we performed a side-by-side comparison of erlotinib and gefitinib effects on a broad spectrum of malignant myeloid cell lines, as well as on primary myeloblasts freshly purified from the bone marrow of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Both erlotinib and gefitinib induce apoptosis of a cell line (KG-1) that represents AML, and differentiation in another cell line (P39) derived from a patient with high-risk MDS. In this setting, erlotinib was more efficient than gefitinib. Erlotinib and gefitinib were equipotent in inducing apoptosis of primary CD34+ myeloblasts from MDS and AML patients, yet had no toxic effect on CD34+ progenitor cells from healthy donors. Although the response of individual MDS and AML patients in vitro was highly heterogeneous, the pro-apoptotic effects of erlotinib and gefitinib correlated significantly. These results suggest that erlotinib and gefitinib share a mechanistically related off-target effect that may be taken advantage of for the therapy of MDS and AML.

Erlotinib exhibits antineoplastic off-target effects in AML and MDS: a preclinical study.

Erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), induces differentiation, cell-cycle arrest, and apoptosis of EGFR-negative myeloblasts of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as in EGFR-negative cell lines representing these diseases (P39, KG-1, and HL 60). This off-target effect can be explained by inhibitory effects on JAK2. Apoptosis induction coupled to mitochondrial membrane permeabilization occurred independently from phenotypic differentiation. In apoptosis-sensitive AML cells, erlotinib caused a rapid (within less than 1 hour) nucleocytoplasmic translocation of nucleophosmin-1 (NPM-1) and p14(ARF). Apoptosis-insensitive myeloblasts failed to manifest this translocation yet became sensitive to apoptosis induction by erlotinib when NPM-1 was depleted by RNA interference. Moreover, erlotinib reduced the growth of xenografted human AML cells in vivo. Erlotinib also killed CD34(+) bone marrow blasts from MDS and AML patients while sparing normal CD34(+) progenitors. This ex vivo therapeutic effect was once more associated with the nucleocytoplasmic translocation of NPM-1 and p14(ARF). One patient afflicted with both MDS and non-small cell lung cancer manifested hematologic improvement in response to erlotinib. In summary, we here provide novel evidence in vitro, ex vivo, and in vivo for the potential therapeutic efficacy of erlotinib in the treatment of high-risk MDS and AML.

Nonapoptotic role for Apaf-1 in the DNA damage checkpoint.

Apaf-1 is an essential factor for cytochrome c-driven caspase activation during mitochondrial apoptosis but has also an apoptosis-unrelated function. Knockdown of Apaf-1 in human cells, knockout of apaf-1 in mice, and loss-of-function mutations in the Caenorhabditis elegans apaf-1 homolog ced-4 reveal the implication of Apaf-1/CED-4 in DNA damage-induced cell-cycle arrest. Apaf-1 loss compromised the DNA damage checkpoints elicited by ionizing irradiation or chemotherapy. Apaf-1 depletion reduced the activation of the checkpoint kinase Chk1 provoked by DNA damage, and knockdown of Chk1 abrogated the Apaf-1-mediated cell-cycle arrest. Nuclear translocation of Apaf-1, induced in vitro by exogenous DNA-damaging agents, correlated in non-small cell lung cancer (NSCLC) with the endogenous activation of Chk-1, suggesting that this pathway is clinically relevant. Hence, Apaf-1 exerts two distinct, phylogenetically conserved roles in response to mitochondrial membrane permeabilization and DNA damage. These data point to a role for Apaf-1 as a bona fide tumor suppressor.

Src kinase inhibitors induce apoptosis and mediate cell cycle arrest in lymphoma cells.

Src kinases are involved in multiple cellular contexts such as proliferation, adhesion, tumor invasiveness, angiogenesis, cell cycle control and apoptosis. We here demonstrate that three newly developed dual selective Src/Abl kinase inhibitors (SrcK-I) (AZM559756, AZD0530 and AZD0424) are able to induce apoptosis and cell cycle arrest in BCR-ABL, c-KIT and platelet-derived growth factor-negative lymphoma cell lines. Treatment of DOHH-2, WSU-NHL, Raji, Karpas-299, HUT78 and Jurkat cells with SrcK-I revealed that the tested substances were effective on these parameters in the cell lines DOHH-2 and WSU-NHL, whereas the other tested cell lines remained unaffected. Phosphorylation of Lyn and in particular Lck were affected most heavily by treatment with the SrcK-I. Extrinsic as well as intrinsic apoptosis pathways were activated and elicited unique expressional patterns of apoptosis-relevant proteins such as downregulation of survivin, Bcl-XL and c-FLIP. Protein levels of c-abl were downregulated and Akt phosphorylation was decreased by treatment with SrcK-I. Basal expression levels of c-Myc were notably lower in sensitive cell lines as compared with nonsensitive cell lines, possibly providing an explanation for sensitivity versus resistance against these novel substances. This study provides the first basis for establishing novel SrcK-I as weapons in the arsenal against lymphoma cells.

The tyrosine kinase inhibitor AMN107 (Nilotinib) exhibits off-target effects in lymphoblastic cell lines.

The aminopyrimidine inhibitor AMN107 (Nilotinib) was rationally designed to antagonize the aberrant tyrosine kinase activity of Bcr-Abl-positive cells. We here evaluated, whether AMN107 is also able to induce apoptosis in Bcr-Abl-negative cells of lymphatic origin. The B-cell lines DOHH-2 and WSU-NHL and the T-cell lines Jurkat and HUT78 were incubated with increasing amounts of AMN107 corresponding to clinically achievable dosages. Subsequently, induced molecular changes were assessed by FACS analysis, Western blot, and enzyme activity assays. Although AMN107 exhibited only a minor apoptosis-inducing effect in the T-cell lines, it exerted a considerable, dose-dependent cytotoxicity in the B-cell lines. Using selective caspase-inhibitors, we show that apoptosis in responder cell lines critically relies on activation of caspase-6 and caspase-9. Cell lines sensitive and resistant towards AMN107 can be discriminated by their differential expression of Src-kinases. Although the AMN107-sensitive cell lines DOHH-2 and WSU-NHL exhibited low or no expression of the Src-kinases Lck, phosphorylated Lck, and Yes with a concomitant high expression of Hck, Lyn, and phosphorylated Lyn, the expression pattern of these kinases was inverse in the AMN107-resistant T-cell lines. In conclusion, this is the first report providing evidence that activity of AMN107 is not restricted to Bcr-Abl, c-Kit, or PDGFR-positive cells, but also extends to lymphatic cell lines of B-cell origin.

Differentiating megakaryocytes in myelodysplastic syndromes succumb to mitochondrial derangement without caspase activation.

Myelodysplastic syndromes (MDS) constitute a preneoplastic condition in which potentially malignant cancer stem cells continuously die during differentiation. This MDS-associated cell death often involves caspase-3 activation, yet can also occur without caspase activation, for instance in differentiating megakaryocytes (MK). We investigated, the mechanisms through which MK from MDS patients undergo premature cell death. While polyploid, mature MK from healthy subjects or MDS patients manifested caspase-3 activation during terminal differentiation, freshly isolated, immature MK from MDS died without caspase-3 activation. Similarly, purified bone marrow CD34(+) cells from MDS patients that were driven into MK differentiation in vitro died without caspase-3 activation at an immature stage, before polyploidization. The premature death of MDS MK was accompanied by the mitochondrial release of cytochrome c, Smac/DIABLO and endonuclease G, a caspase-independent death effector, as well loss of the mitochondrial membrane potential and plasma membrane phosphatidylserine exposure before definitive loss of viability. Thus, a stereotyped pattern of mitochondrial alterations accompanies differentiation-associated MK death in MDS.

The molecular biology of TRAIL-mediated signaling and its potential therapeutic exploitation in hematopoietic malignancies.

Tumor necrosis factor apoptosis ligand (TRAIL) is a type II membrane-bound ligand displaying expression in a broad range of tissues and exhibiting a high grade of homology with the cytotoxic Fas ligand. Interest in TRAIL grew after evidence emerged, that induction of TRAIL-mediated signaling destroyed malignant cells while sparing normal cells. Employing the extrinsic pathway of apoptosis, TRAIL-stimulation is characterized by initial adaptor recruitment and the subsequent activation of caspases. Besides promoting apoptosis, stimulation of the TRAIL receptors may also activate survival signals via the transcription factor NF-kappaB. Moreover, evaluation of the physiological roles of TRAIL-mediated signaling pathways provides evidence for a regulatory function within the immune system. Thus a complex picture of TRAIL-mediated signaling evolves, underscoring the necessity to define its modes of action while assessing its therapeutic potential. This review outlines the current knowledge on the physiological role of TRAIL and discusses its therapeutic potential with particular focus on malignancies of the hematopoietic system.

Prostate-apoptosis-response-gene-4 increases sensitivity to TRAIL-induced apoptosis.

The capacity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to preferentially induce apoptosis in malignant cells while sparing normal tissues renders it an attractive therapeutic agent. Nevertheless, the molecular determinants governing sensitivity towards TRAIL remain to be defined. Acknowledging the previously demonstrated deregulation of prostate-apoptosis-response-gene-4 (par-4) in ex vivo cells of patients suffering from acute and chronic lymphatic leukemia, we here tested the hypothesis that expression of par-4 influences sensitivity to TRAIL. Evaluating this hypothesis we show, that par-4-transfected T-lymphoblastic Jurkat cells exhibit a considerably increased rate of apoptosis upon incubation with an agonistic TRAIL-antibody as compared to their mock-transfected counterparts. Defining the underlying molecular mechanisms we provide evidence, that par-4 enhances sensitivity towards TRAIL by employing crucial members of the extrinsic pathway. Thus, par-4-overexpressing Jurkat clones show an enforced cleavage of c-Flip(L) together with an increased activation of the initiator caspases-8 and -10. In addition, expression of par-4 enables cells to down-regulate the inhibitor-of-apoptosis proteins cIAP-1, cIAP-2, XIAP and survivin with a concomitantly enhanced activation of the executioner caspases-6 and -7. Supporting the crucial role of caspase-8 in par-4-promoted apoptosis we demonstrate that inhibition of caspase-8 considerably reduces TRAIL-induced apoptosis in par-4 and mock-transfected Jurkat clones and reverses the described molecular changes. In conclusion, we here provide first evidence that expression of par-4 in neoplastic lymphocytes augments sensitivity to TRAIL-induced cell death and outline the responsible molecular mechanisms, in particular the crucial role of caspase-8 activation.

Novel agents aiming at specific molecular targets increase chemosensitivity and overcome chemoresistance in hematopoietic malignancies.

In hematologic neoplasias primary or secondary resistance of malignant cells towards the applied treatment presents the major clinical obstacle in the induction of remission and definite cure. Evaluation of the underlying molecular mechanisms determining response or resistance not only enables the clinician to define prognostic markers, but moreover facilitates the design of molecularly targeted agents potentially reversing the causative lesion. Deregulation of apoptosis is considered to contribute to the emergence and propagation of the malignant clone, and several molecular alterations hindering programmed cell death and thus leading to chemoresistance have been defined. While reviewing these molecular alterations this article moreover focuses on the impact of new therapeutic agents, which specifically exploit the knowledge of the molecular characteristics of malignant hematopoetic cells.