CDC37 as a novel target for the treatment of NPM1-ALK expressing anaplastic large cell lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive CD30+ T-cell lymphoma that accounts for 2-8% and 10-15% of non-Hodgkin lymphomas in adults and children, respectively. The currently used standard therapy for anaplastic lymphoma kinase (ALK, a member of the insulin receptor superfamily) positive ALCL has limited effectiveness, resulting in a substantial percentage of cases with poor outcomes, either failing to enter remission or relapse within a few months after starting treatment. Thus, there is a clear unmet clinical need for developing novel, effective and safer therapeutic strategies for ALCL. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Approximately 50% of ALCL cases are positive for the NPM1-ALK fusion chimera generated by the t(2;5) chromosomal translocation. The oligomerization domain of NPM1 in the fusion protein NPM1-ALK mediates the ligand-independent dimerization of chimeric protein, which results in constitutive activation of the chimeric tyrosine kinase activity leading to downstream signaling pathways responsible for the oncogenicity. As NPM1-ALK is an established heat shock protein 90 (HSP90) client protein, we hypothesized that disruption of the interaction between master chaperone HSP90 and co-chaperone Cdc37 would be a viable druggable target. Celastrol is a natural compound extracted from the root bark of Chinese medicinal plant Tripterygium wilfordi. Anticancer properties of celastrol are attributed to its action on the interaction of HSP90 and its co-chaperone Cdc37. Hsp90 is an ATP-dependent master molecular chaperone. Cdc37 is a Hsp90 co-chaperone and disruption of Hsp90-Cdc37 interaction will result in the degradation of Hsp90 client proteins. Celastrol disrupts the interaction of Hsp90 and Cdc37 through inhibition of Hsp90 ATPase activity without blockage of ATP binding site as observed with Hsp90 inhibitors. We used NPM1-ALK endogenously expressing Karpas299, SUDHL-1 and ectopically expressing Ba/F3-FG-NPM1-ALK cell lines in our study. Treatment with celastrol (0.1-1 µM) significantly induced apoptosis dose dependently in ALCL cells. Apoptosis was associated with activated caspase 3 and poly (ADP-ribose) polymerase cleavage. We also observed upregulation of proapoptotic protein BAD and downregulation of antiapoptotic proteins survivin and MCL1. Celastrol treatment inhibited NPM1-ALK mediated signaling and downstream signaling effectors STAT3, AKT, and ERK1/2 by degradation of the NPM1-ALK fusion protein. Cell survival was evaluated by clonogenic survival assay and results were clearly indicative that the clonogenic survival is diminished in celastrol treated samples compared to controls. Our immunoprecipitation studies clearly demonstrated that celastrol treatment disrupted the interaction between Hsp90 and Cdc37 and induced degradation of the NPM1-ALK fusion protein. Altogether, targeting Cdc37 using celastrol is a novel therapeutic approach to induce apoptosis in ALCL cells expressing NPM1-ALK and warrants developing future therapeutic intervention strategies. Disclosures Ganguly: Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

CDC37 as a novel target for the treatment of NPM1-ALK expressing anaplastic large cell lymphomas Sudhakiranmayi Kuravi 1 , Elizabeth Parrott 2 , Giridhar Mudduluru 1 , Janice Cheng 1 , Siddhartha Ganguly 1 , Yogen Saunthararajah 3 , Roy A. Jensen 4 , Brian S. Blagg 5 , Joseph P. McGuirk 1 and Ramesh Balusu 6,7 Anaplastic large cell lymphoma (ALCL) represents a rare and aggressive subtype of CD30-positive peripheral T-cell lymphoma, which accounts for 5-10% of non-Hodgkin lymphomas in adults and 10-30% in children 1 . Anaplastic lymphoma kinase (ALK) fusions are present in both solid and hematologic malignancies. More than 80% of ALK-positive ALCLs are hallmarked by the fusion gene nucleophosmin (NPM1)-ALK generated by the t(2;5) chromosomal translocation, and 5% of non-small cell lung cancer patients carry echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion 1 . NPM1 is a nucleolar phosphoprotein involved with chaperoning of proteins and nucleic acids 2 . ALK is a receptor tyrosine kinase belonging to the insulin receptor superfamily. NPM1-ALK fusion protein p80 is derived from the fusion of the N-terminal oligomerization domain of NPM1 (1-110 aa) and the C-terminal tyrosine kinase domain of ALK (1058-1620 aa) 3 . In these malignancies, the homodimerization of NPM1-ALK leads the constitutive activation of a fusion kinase in a ligand-independent manner. The persistently active tyrosine kinase NPM1-ALK triggers multiple intracellular downstream signaling pathways including AKT, ERK1/2, and STAT3, which results in proliferation and survival of ALCL cells 1 .
Many ALK tyrosine kinase inhibitors have been developed, evaluated, and approved for clinical trials in malignancies associated with ALK fusion genes. Clinical resistance is a major problem associated with these ALK inhibitors. One of the explored mechanisms responsible for resistance to ALK inhibitors in these malignant cells is through acquired secondary mutations in the kinase domain 4 . Overall, current therapeutic approaches used for the treatment of ALK-positive ALCLs has limited effectiveness, resulting in a substantial percentage of cases with poor outcomes, either failing to achieve remission or relapsing within a short period. Hence, there is a need to focus on developing novel and effective treatment strategies to overcome this clinical conundrum.
More than 500 protein kinases are present in the human kinome and they play an essential role in cellular processes such as cell proliferation, signaling, differentiation, and apoptosis 5 . Dysregulation of these protein kinase functions (genetic alterations including mutations or fusion genes) is responsible for various pathological conditions including cancer. Protein kinases depend on a central molecular chaperone, heat shock protein 90 (HSP90) for their maturation and to protect against proteasomal degradation. HSP90 is an ATPase-dependent master chaperone that accounts for around 10% of the proteome. It is important to note, the majority of mutated kinase proteins involved with malignancies are maintained through HSP90 dependency, leading to "HSP90 addiction." Exploitation of HSP90 by oncogenic kinases for their stability and maturation makes HSP90 as a viable molecular target 6 . Several HSP90 inhibitors bind to the ATP binding site of HSP90 and promote degradation of oncogenic protein kinases. Mutant fusion kinases such as BCR-ABL and NPM1-ALK are known HSP90 client proteins in hematologic malignancies 7 . Several preclinical studies have been very supportive in considering HSP90 as a therapeutic target. However, many clinical trials using HSP90 inhibitors have been halted due to toxicity associated with the robust heat shock response 8 . An alternative approach to effectively target these HSP90dependent oncogenic kinases, while minimizing nonspecific toxicity could be disrupting the interaction between HSP90 and its co-chaperones. HSP90 chaperone machinery is complex, and along with HSP90, many cochaperones are involved in the process. Cyclin-dependent kinase 37 (CDC37) is a specific co-chaperone recruiter for a diverse group of protein kinases. HSP90-mediated maturation of these oncogenic kinases strictly depends on CDC37 9 . Celastrol, a triterpene molecule extracted from the Chinese herbal plant Tripterygium wilfordii Hook F, blocks the interaction between HSP90 and CDC37 and thereby triggers the degradation of its dependent client protein kinases. Mechanistically, celastrol does not interfere with ATP binding to HSP90, but inhibits the critical interaction and binding between the N-terminal region of CDC37 (Arg167) and the middle domain of HSP90 (Glu33) 10 .
In this study, we tested the fusion oncogene NPM1-ALK dependency on co-chaperone CDC37 by disrupting the interaction between HSP90 and CDC37, using celastrol as a therapeutic approach in ALCL cells. We have utilized a total of six cell lines: NPM1-ALK endogenously expressing human ALCL cell lines (SUDHL-1, Karpas-299, SUP-M2, SR-786, and DEL), and our laboratory generated ectopically overexpressing Ba/F3-FG-NPM1-ALK, a murine cell line. In this report, we present celastrol-mediated effects on apoptosis, proliferation, oncogenic signaling, and CD30 (cluster of differentiation 30) expression in ALCL cells.
Earlier studies demonstrated NPM1-ALK as an HSP90 client protein by using the HSP90 ATPase inhibitor-17AAG 11 . Several protein kinases are well characterized for their dependency on CDC37 co-chaperoning, but very limited studies are available for fusion kinases. In hematologic malignancies, BCR-ABL was the first fusion kinase identified to be dependent on CDC37 co-chaperone interaction for its stability 12 . Our experiments confirmed that endogenous NPM1-ALK fusion protein levels in SUDHL-1, Karpas-299, SUP-M2, SR-786, and DEL cells were diminished with celastrol treatment in a dosedependent manner (0.25-1.0 µM) after 24 h (Fig. 1a). In similar lines, ectopically overexpressed NPM1-ALK was also downregulated in Ba/F3 cell line. The decrease in total NPM1-ALK resulted in a reduction of active phosphorylated NPM1-ALK (Fig. 1a). With the significant decrease in protein levels of total NPM1-ALK and phospho-NPM1-ALK, we further examined the influence of these effects on relevant NPM1-ALK downstream signaling in five NPM1-ALK expressing cell lines. AKT/ PI3K, MAPK/ERK, and STAT3 are well-studied survival signaling pathways that are activated by NPM1-ALK in CD30-positive ALCL cells 13 . Celastrol-mediated downregulation of NPM1-ALK phosphorylation inhibited downstream signaling activators phosphorylated AKT, ERK1/2, and STAT3 in a dose-dependent manner. There was a minimal effect on total AKT, ERK1/2, STAT3 proteins, and β-actin levels were used as loading control (Fig. 1b). Based on these experimental results, celastrol downregulates fusion protein NPM1-ALK by blocking the interaction between HSP90 and CDC37, which in turn inhibits downstream survival signaling cascade AKT, ERK1/2, and STAT3.
We then evaluated the ability of celastrol to induce apoptosis in NPM1-ALK endogenously expressing SUDHL-1, Karpas-299, SUP-M2, SR-786, DEL, and ectopically expressing Ba/F3-FG-NPM1-ALK lymphoma cell lines along with normal T cells. All selected ALCL cell lines were treated with celastrol (0.1-1.0 µM) for 48 h, and apoptosis was measured by flow cytometry using FITC-annexin V and TO-PRO-3. All of the tested NPM1-ALK fusion gene expressing cell lines were sensitive to celastrol and showed induced apoptosis in a dosedependent manner compared to controls but no significant effect on normal T cells (Fig. 2a). Overall, celastrol showed growth inhibitory effects on both endogenous and ectopic NPM1-ALK expressing cell lines. PARP (poly (ADP-ribose) polymerase) catalyzes poly(ADP-ribosyl) ation of nuclear proteins involved in DNA transcription, replication, and repair. During apoptosis, it is well known that PARP is cleaved by specific caspases. Cancer cells are associated with an imbalance between pro-and antiapoptotic genes 14 . Therefore, we examined PARP cleavage, activation of caspases, and differential regulation of pro-apoptotic (BAX) and anti-apoptotic molecules (survivin, Bcl2, and c-Myc). Two cell lines, SUDHL-1 and Karpas-299 were treated with 0.25-1.0 µM celastrol for 24 h. Celastrol treatment in these cell lines showed PARP cleavage, downregulation of procaspases 8 and 9, upregulation of pro-apoptotic protein BAX and downregulation of anti-apoptotic proteins survivin, Bcl2, and c-Myc in a dose-dependent manner (Fig. 2b). All the evaluated proteins involved in apoptosis are activated by the downstream signaling axis of NPM1-ALK 13 .
The chimera NPM1-ALK drives the proliferation of ALCL cells. Therefore, we tested the effect of celastrol on the proliferation of NPM1-ALK-positive cells using a standard methylcellulose clonogenic assay. Karpas-299 and SUP-M2 cells were treated with 0, 0.5, and 1.0 µM celastrol for 24 h. Cells were then washed, mixed with a MethoCult medium, plated, and incubated for 8 days. The total number of colonies were counted in each condition. There was a significant reduction in the clonogenic potential of celastroltreated Karpas-299 and SUP-M2 cells compared to control due to inhibition of NPM1-ALK activation (Fig. 2c).
ALCL cells are immunophenotypically characterized by the strong expression of the CD30 marker, a member of the tumor necrosis factor (TNF) receptor family, which is transcriptionally upregulated through the NPM1-ALKmediated ERK1/2 and STAT3 pathways 15 . CD30 activation contributes to lymphoma cell proliferation through activated NF-κB and other anti-apoptotic mechanisms. We analyzed the effect of NPM1-ALK downregulation on CD30 expression in Karpas-299 and SUDHL-1 cells (Fig.  2d). ALCL cell lines were treated with celastrol and analyzed for CD30 expression by flow cytometry. The results showed treatment with celastrol in the SUDHL-1 cell line exhibited a higher response in the reduction of CD30 in comparison to Karpas-299 cell line. The differential response might be due to the variable expression levels of NPM1-ALK and downstream signaling effector molecules in these cell lines.
In summary, our results show for the first time that inhibition of the interaction between CDC37 and HSP90 using celastrol represents a novel therapeutic approach in ALCL cells expressing the NPM1-ALK fusion gene. These observations further pave the way to consider CDC37 as a novel molecular target for the treatment of NPM1-ALK expressing ALCL cells and warrants developing future therapeutic intervention strategies. Fig. 1 Celastrol downregulates NPM1-ALK fusion protein and its signaling: (a) celastrol treatment depletes NPM1-ALK protein levels and inhibits activation of NPM1-ALK fusion kinase. NPM1-ALK expressing SUDHL-1, Karpas-299, SUP-M2, SR-786, DEL, and Ba/F3-FG-NPM1-ALK cells were treated with indicated concentrations of celastrol for 24 h. At the end of the treatment period, cell lysates were made, and immunoblot analyses were performed for total NPM1-ALK and phospho-NPM1-ALK proteins. b Depletion of NPM1-ALK leads to inhibition of downstream survival signaling cascade. NPM1-ALK expressing ALCL cell lines treated with celastrol and western blot analyses were performed for downstream effector molecules pSTAT3, pAKT, pERK1/2 along with total proteins. β-actin served as the loading control