Identification of heat shock protein 32 (Hsp32) as a novel survival factor and therapeutic target in neoplastic mast cells

Abstract

Systemic mastocytosis (SM) is a myeloid neoplasm characterized by increased survival and accumulation of neoplastic mast cells (MCs). In most patients, the D816V-mutated variant of KIT is detectable. We report here that heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a novel KIT-inducible survival factor in neoplastic IntroductionSystemic mastocytosis (SM) is a myeloid neoplasm characterized by increased survival and accumulation of neoplastic mast cells (MCs) in internal organs. [1][2][3][4][5] Indolent and aggressive variants of SM have been described. [1][2][3][4][5][6][7] In patients with aggressive SM (ASM) or MC leukemia (MCL), the response to conventional drugs is poor, and the prognosis is grave. [4][5][6][7] These patients are candidates for cytoreductive or experimental therapy. [4][5][6][7][8][9][10] Indeed, several attempts have been made to identify novel therapeutic targets in neoplastic MCs. 4,5,[8][9][10] In most patients with SM, including those with ASM or MCL, the KIT mutation D816V is detectable. [5][6][7][11][12][13][14][15] This mutation is associated with ligand-independent activation of KIT. 16 Therefore, a number of attempts have been made to identify drugs interfering with the tyrosine kinase (TK) activity of KIT D816V. 9,10,[17][18][19][20][21] One of these drugs is PKC412, which counteracts in vitro growth of neoplastic MCs harboring KIT D816V. 17,18 In addition, PKC412 was found to inhibit growth of neoplastic MCs in a patient with MCL. 19 However, despite its impressive effects, PKC412 alone may not be sufficient to induce long-lasting complete responses in ASM/MCL. 19 Therefore, current research focuses on additional drug targets in neoplastic MCs and respective targeted drugs. 9 One attractive type of target are the survival factors expressed in neoplastic MCs.Heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a stress-related survival factor [22][23][24] that is expressed constitutively in various neoplastic cells. [25][26][27][28][29][30][31] Thus, whereas previous studies have pointed to the protective role of Hsp32 in mesenchymal cells in inflammatory reactions and the related stress response, [22][23][24] more recent data suggest that neoplastic cells also use Hsp32 as a survival-related molecule. [25][26][27][28][29][30][31] Moreover, Hsp32-targeting drugs have been described as counteracting in vivo growth of experimental tumors in mice. [25][26][27] We have recently shown that Hsp32/HO-1 is constitutively expressed in neoplastic cells in chronic myeloid leukemia (CML), and that the CMLspecific oncoprotein BCR-ABL promotes expression of Hsp32. 29 With regard to MCs, little is known about the expression of Hsp32. In rats, normal tissue MCs express low baseline levels of Hsp32, and its expression may increase after cell activation. 32 It has also been shown that activated rat MCs can use Hsp32 as a cytoprotective survival factor. 32 So far, however, expression of Hsp32 has not been examined in the context of mastocytosis. In the...

Polo-like kinase-1 as a novel target in neoplastic mast cells: demonstration of growth-inhibitory effects of small interfering RNA and the Polo-like kinase-1 targeting drug BI 2536

“…In the second patient, MC were purified to homogeneity (purity >98%) by cell sorting as described elsewhere. 39 Primers for human Plk-1 were: 5´-CCCATCTTCTGGGTCAGCAAG-3´ (forward) and 5´-AAGAG-CACCCCC ACGCTGTT-3´ (reverse). The polymerase chain reaction (PCR) conditions were: annealing 30 s (65°C); extension, 1 min (72°C); denaturation 30 s (94°C); 30 cycles.…”

“…Equal loading was confirmed by determining β-actin mRNA levels using published primers. 39 For quantitative PCR, cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and random primers (both from Invitrogen Inc, Carlsbad, CA, USA) according to the manufacturer's instructions. The primers used were: Plk-1: 5´-CCTCCGGATCAAGAAGAATGAA-3´ (forward) and 5´-GCAGTGGGATCTGT CTGAAGCA-3` (reverse); Abl: 5`-TCTATGATTTTGTGGCCAGTGGAG-3` (forward) and 5`-GCCTAAGACCCGGAGCTTTTCA-3` (reverse).…”

“…39 The mouse monoclonal antibody K50-483 (work dilution 1:20) from Becton Dickinson Pharmingen (San Jose, CA, USA) and a biotinylated goat-anti-mouse IgG antibody (Biocare, San Diego, CA, USA) were applied for detection of phosphorylated Plk-1 (pPlk-1), whereas for detection of total Plk-1, a polyclonal rabbit anti-Plk-1 antibody (1:20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and biotinylated goat-anti-rabbit IgG (Biocare) were employed. Slides were incubated with anti-Plk-1 antibodies overnight.…”

“…44 To confirm apoptosis, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed as reported previously 39,45 using HMC-1 cells and C2 cells incubated with BI 2536 (100 nM) or control medium for 48 h. After fixation (with formaldehyde and 70% ethanol) and staining, cells were washed and analyzed with a Nikon Eclipse E 800 fluorescence microscope (Tokyo, Japan). For evaluation of caspase cleavage, HMC-1 cells were incubated with BI 2536 (1-100 nM) or control medium for 48 h. Western blotting was performed essentially as described elsewhere 39,43,45 using a polyclonal antibody against cleaved caspase-3, monoclonal antibody 18C8 against cleaved caspase-8, and a polyclonal antibody against cleaved caspase-9 (all from Cell Signaling Technology, Danvers, MA, USA). A polyclonal antibody against β-actin (Sigma) was applied to confirm equal loading.…”

Targeted therapy inKITD816V-positive mastocytosis: waiting for proof-of-principle

“…However, the kinase domain KIT mutation found in virtually all patients with SM (i.e. KITD816V) is IM-resistant, but in vitro sensitivity has been demonstrated using dasatinib [7,13], nilotinib (although others have shown lack of activity) [8,9], MLN518 [10], PKC412 [11], EXEL-0862 [12], triptolide (a Chinese herb-derived diterpenoid) [14], heat shock protein 32-targeting drugs [15], and APcK110 (a novel KIT inhibitor) [16].…”