Virginia M. Goettl

Selective inhibitors of nuclear export show that CRM1/XPO1 is a target in chronic lymphocytic leukemia

The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the Eμ-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies.

The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

17-DMAG targets the nuclear factor-κB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition

The HSP90 client chaperone interaction stabilizes several important enzymes and antiapoptotic proteins, and pharmacologic inhibition of HSP90 results in rapid client protein degradation. Therefore, HSP90 inhibition is an attractive therapeutic approach when this protein is active, a phenotype commonly observed in transformed but not normal cells. However, preclinical studies with HSP90 inhibitors such as 17-AAG demonstrated depletion of only a subset of client proteins and very modest tumor cytotoxicity in chronic lymphocytic leukemia (CLL) cells. Herein, we describe another HSP90 inhibitor, 17-DMAG, which is cytotoxic to CLL but not normal lymphocytes. Treatment with 17-DMAG leads to depletion of the HSP90 client protein IKK, resulting in diminished NF-kappaB p50/p65 DNA binding, decreased NF-kappaB target gene transcription, and caspase-dependent apoptosis. Furthermore, treatment with 17-DMAG significantly decreased the white blood cell count and prolonged the survival in a TCL1-SCID transplant mouse model. The ability of 17-DMAG to function as an NF-kappaB inhibitor is of great interest clinically, as few currently available CLL drugs target this transcription factor. Therefore, the effect of 17-DMAG on NF-kappaB signaling pathways represents a novel therapy warranting further clinical pursuit in this and other B-cell lymphoproliferative disorders.

Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Brutons tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Glutamate uptake is attenuated in spinal deep dorsal and ventral horn in the rat spinal nerve ligation model

Alteration of glutamatergic (GLU) neurotransmission within the spinal cord contributes to hyperalgesic and allodynic responses following nerve injury. In particular, changes in expression and efficacy of glutamate transporters have been reported. Excitatory, pain transmitting primary afferent neurons utilizing glutamate as an excitatory neurotransmitter project to both superficial (I-II) and deep (III-V) laminae of the dorsal horn. These experiments were designed to examine changes in glutamate uptake occurring concomitantly within the spinal deep dorsal and ventral horn in situ after experimentally induced neuropathic pain. In vivo voltammetry, using microelectrode arrays configured for enzyme-based detection of GLU were employed. Sprague-Dawley rats had either sham surgery or tight ligation of L5 and L6 spinal nerves (SNL). Four to six weeks later, the L4-L6 spinal cord of chloral hydrate-anesthetized animals was exposed, and ceramic-based glutamate microelectrodes equipped with glass micropipettes 50 microm from the recording surfaces were placed stereotaxically at sites within the spinal cord. Pressure ejection of GLU into the ipsilateral L5-L6 spinal cord resulted in a 72% reduction of GLU uptake in SNL rats compared to sham controls in the ipsilateral L5-L6 deep dorsal horn and a 96% reduction in the ventral horn. In contrast, in the same animals, the contralateral L5-L6 or the ipsilateral L4 spinal cord showed no change in glutamate uptake. The data suggest that spinal nerve ligation produced attenuated glutamate uptake activity extending into the deep dorsal and ventral horn. The study suggests that plasticity related to spinal nerve injury produces widespread alteration in glutamate transporter function that may contribute to the pathophysiology of neuropathic pain.

Reduced basal release of serotonin from the ventrobasal thalamus of the rat in a model of neuropathic pain.

&NA; Drugs that inhibit reuptake of monoamines are frequently used to treat pain syndromes, e.g. neuropathy or fibromyalgia, where mechanical allodynia is present. Several lines of evidence suggest the involvement of supraspinal sites of action of these drugs. However, a direct study of supraspinal serotonin (5‐HT) or norepinephrine (NE) release in an animal model in which allodynia is expressed, e.g. neuropathy, has not been done. The ventrobasal (VB) thalamus and the hypothalamus are major supraspinal projection regions for spinal neurons that transmit nociceptive information and are innervated by monoaminergic fibers. This study determined if peripheral neuropathy would induce changes in extracellular monoamines in VB thalamus and hypothalamus. Male Sprague–Dawley rats had spinal nerve roots L5 and L6 tightly ligated (neuropathic rats; NP) or sham (SHAM) surgery; contralateral and ipsilateral VB thalamus and contralateral hypothalamus were dialyzed with modified artificial cerebral spinal fluid (aCSF), with and without fluoxetine. NP rats had significantly decreased 5‐HT content in dialysates of the contralateral VB thalamus compared with SHAM rats with (82% decrease) or without (63% decrease) fluoxetine in the perfusion medium over the 180 min of the study. There were no differences in the ipsilateral VB thalamus. In contrast, release of 5‐HT was unchanged in the hypothalamic dialysates of SHAM vs. NP rats. NE release was not different in dialysates of either the VB thalamus or hypothalamus of SHAM vs. NP rats. Synthesis of 5‐HT, as assessed by accumulation of 5‐hydroxytrytophan after treatment with an L‐amino acid decarboxylase inhibitor, was not different between NP and SHAM rats in VB thalamic and hypothalamic brain tissue. This study is the first to demonstrate changes in monoamine release supraspinally in NP rats. The differential effect between VB thalamus and hypothalamus suggests that a terminal field change may be involved. Putative mechanisms for mediating this change include alterations of GABA‐ergic systems and/or plasticity related to alterations in N‐methyl‐D‐aspartate receptor activation and nitric oxide release related to afferent hyperactivity induced by neuropathic pain.

Selinexor suppresses Downstream Effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells

Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells

Next-generation XPO1 inhibitor shows improved efficacy and in vivo tolerability in hematological malignancies

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.

Sciatic nerve axotomy in aged rats: response of motoneurons and the effect of GM1 ganglioside treatment.

The number, size, and staining intensity of choline acetyltransferase (ChAT)-immunopositive cells in the retrodorsal lateral nucleus (RDLN) of the spinal cord were studied in young (3-5 months old) and aged (22-24 months old) rats following left sciatic nerve distal transection (axotomy) and treatment with GM1 ganglioside. The cell size and the ChAT immunostaining density were decreased in the RDLN of non-manipulated as well as in the contralateral intact side of axotomized aged rats. Axotomy had no effect on the number of RDLN motoneurons in both aged and young rats. In the young rats, there was a decrease in the size of motoneurons 7 days post-axotomy and a partial spontaneous recovery occurred by 21 days. Axotomy did not reduce further the size of aged motoneurons, however. The ChAT staining intensity of the axotomized RDLN declined in both age groups after 7 days, and there was spontaneous near normal recovery by 21 days. In the aged rats, GM1 administration for 7 days corrected the cell size and ChAT immunoreactivity of the contralateral intact RDLN. With regard to axotomized RDLN neurons, 7 days of GM1 restored the cell size but not the ChAT immunostaining in young animals. The same treatment schedule, however, corrected both cell size and staining in aged rats. Administration of GM1 for 21 days had no further effect on the morphometric parameters of the axotomized motoneurons in aged rats, but slightly enhanced the recovery of ChAT immunostaining in young rats. Thus, it appears that GM1 facilitates the phenotypic recovery of RDLN motoneurons during aging and after axotomy.

GM1 enhances dopaminergic markers in the brain of aged rats

A number of presynaptic markers are compromised in the dopaminergic neurons of aged Sprague-Dawley rats (22 months old) compared with young rats (3 months old). Indeed, in the striatum of the aged rats there is a diminished capacity to transport dopamine (DA), to bind the dopamine transporter (DAT) marker mazindol, to bind the vesicular monoamine transporter 2 (VMAT2) marker dihydrotetrabenazine, and to release DA under basal conditions or after induction by K(+) or amphetamine. Furthermore, the expression of DAT and VMAT2 mRNA in the midbrain is suppressed. GM1 ganglioside, 30 mg/kg ip daily, administered for 30 days, restores the afore-mentioned markers to values approaching those for young rats. Taken together with our published observations that GM1 partially restores tyrosine hydroxylase activity and DA metabolism in aged nigrostriatal and mesoaccumbal neurons and improves their morphology, our work suggests that GM1 might act as a dopaminergic neurotrophic factor in the aged brain and be a useful adjuvant for treating age-associated dopaminergic deficits.