Bandana Sharma

The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.

The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT

The crizotinib-resistant ALKF1174L mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALKF1174L. Here we demonstrate acquired resistance to TAE684 and LDK378 in ALKF1174L-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALKF1174L-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance.

Mutations that disrupt PHOXB interaction with the neuronal calcium sensor HPCAL1 impede cellular differentiation in neuroblastoma

Heterozygous germline mutations in PHOX2B, a transcriptional regulator of sympathetic neuronal differentiation, predispose to diseases of the sympathetic nervous system, including neuroblastoma and congenital central hypoventilation syndrome (CCHS). Although the PHOX2B variants in CCHS largely involve expansions of the second polyalanine repeat within the C-terminus of the protein, those associated with neuroblastic tumors are nearly always frameshift and truncation mutations. To test the hypothesis that the neuroblastoma-associated variants exert their effects through loss or gain of protein–protein interactions, we performed a large-scale yeast two-hybrid screen using both wild-type (WT) and six different mutant PHOX2B proteins against over 10 000 human genes. The neuronal calcium sensor protein HPCAL1 (VILIP-3) exhibited strong binding to WT PHOX2B and a CCHS-associated polyalanine expansion mutant but only weakly or not at all to neuroblastoma-associated frameshift and truncation variants. We demonstrate that both WT PHOX2B and the neuroblastoma-associated R100L missense and the CCHS-associated alanine expansion variants induce nuclear translocation of HPCAL1 in a Ca2+-independent manner, while the neuroblastoma-associated 676delG frameshift and K155X truncation mutants impair subcellular localization of HPCAL1, causing it to remain in the cytoplasm. HPCAL1 did not appreciably influence the ability of WT PHOX2B to transactivate the DBH promoter, nor did it alter the decreased transactivation potential of PHOX2B variants in 293T cells. Abrogation of the PHOX2B–HPCAL1 interaction by shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B led to impaired neurite outgrowth with transcriptional profiles indicative of inhibited sympathetic neuronal differentiation. Our results suggest that certain PHOX2B variants associated with neuroblastoma pathogenesis, because of their inability to bind to key interacting proteins such as HPCAL1, may predispose to this malignancy by impeding the differentiation of immature sympathetic neurons.

Cbx3/HP1γ deficiency confers enhanced tumor-killing capacity on CD8+ T cells

Cbx3/HP1γ is a histone reader whose function in the immune system is not completely understood. Here, we demonstrate that in CD8+ T cells, Cbx3/HP1γ insufficiency leads to chromatin remodeling accompanied by enhanced Prf1, Gzmb and Ifng expression. In tumors obtained from Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells, there is an increase of CD8+ effector T cells expressing the stimulatory receptor Klrk1/NKG2D, a decrease in CD4+ CD25+ FOXP3+ regulatory T cells (Treg cells) as well as CD25+ CD4+ T cells expressing the inhibitory receptor CTLA4. Together these changes in the tumor immune environment may have mitigated tumor burden in Cbx3/HP1γ-insufficient mice or wild type mice treated with Cbx3/HP1γ-insufficient CD8+ T cells. These findings suggest that targeting Cbx3/HP1γ can represent a rational therapeutic approach to control growth of solid tumors.

Overcoming Resistance to the THZ Series of Covalent Transcriptional CDK Inhibitors

Irreversible inhibition of transcriptional cyclin-dependent kinases (CDKs) provides a therapeutic strategy for cancers that rely on aberrant transcription; however, lack of understanding of resistance mechanisms to these agents will likely impede their clinical evolution. Here, we demonstrate upregulation of multidrug transporters ABCB1 and ABCG2 as a major mode of resistance to THZ1, a covalent inhibitor of CDKs 7, 12, and 13 in neuroblastoma and lung cancer. To counter this obstacle, we developed a CDK inhibitor, E9, that is not a substrate for ABC transporters, and by selecting for resistance, determined that it exerts its cytotoxic effects through covalent modification of cysteine 1039 of CDK12. These results highlight the importance of considering this common mode of resistance in the development of clinical analogs of THZ1, identify a covalent CDK12 inhibitor that is not susceptible to ABC transporter-mediated drug efflux, and demonstrate that target deconvolution can be accomplished through selection for resistance.

Abstract PR04: Targeting super-enhancer driven oncogene transcription through cyclin-dependent kinase inhibitors

Cyclin-dependent kinases (CDKs) with primary roles in transcription regulation are emerging as tractable therapeutic targets in cancers driven by the aberrant expression of oncogenic transcription factors. Our goal is to disrupt the myriad and pleomorphic features of oncogenic MYC through inhibiting CDKs involved in its transcriptional amplifier role. CDK7 participates in transcription initiation by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II and also functions as a CDK-activating kinase, while CDK12 functions in transcription elongation and RNA processing. Using a novel covalent CDK7 inhibitor, THZ1, we demonstrated striking activity and selectivity in neuroblastoma (NB) cells driven by high MYCN expression. This response translated to significant tumor regression in a mouse model of high-risk NB, without introducing discernible toxicity. We determined that this effect was associated with global inhibition of MYCN-dependent transcriptional amplification. THZ1 led to preferential downregulation of Pol II occupancy at super-enhancer-associated genes, including MYCN and other master transcription factors critical to neuronal development such as PHOX2B, GATA2, and DBH. Similarly, inhibition of CDK12 activity using a novel, first-in-class small molecule inhibitor, THZ-5-31-1, resulted in potent antitumor activity in MYCN-overexpressing NB cells. THZ-5-31-1 doses sufficient to induce PARP cleavage did not lead to significant inhibition of global transcription elongation. Rather, cytotoxicity was associated with preferential downregulation of RNA processing factors and a higher percentage of immature mRNA transcripts. Together, these results suggest that transcriptional CDK inhibitors, by affecting different aspects of the transcription machinery, may inhibit the growth of cancers driven by oncogenic transcription factors such as MYC. Citation Format: Malgorzata Krajewska, Nathan F. Moore, Edmond Chipumuro, Tinghu Zhang, Eugenio Marco, Clark Hatheway, Bandana Sharma, Nicholas Kwiatkowski, Guo-Cheng Yuan, Richard A. Young, Nathanael S. Gray, Rani E. George. Targeting super-enhancer driven oncogene transcription through cyclin-dependent kinase inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR04.

Abstract LB-C24: Resistance to ALK inhibition is associated with enhanced BRD4 dependence in ALK-mutated MYCN-amplified neuroblastoma

Despite substantial therapeutic advances, the long-term survival rate of patients with high-risk neuroblastoma (NB), a tumor of the peripheral sympathetic nervous system is still dismal. Activating mutations in the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase represent promising therapeutic targets in this disease and have prompted clinical trials of ALK inhibitors. But as with all molecularly targeted therapies, resistance is inevitable and will limit the ultimate utility of this therapeutic strategy. To elucidate the mechanisms of resistance to ALK inhibitors, we have generated isogenic NB cells, expressing the tumorigenic ALKF1174L mutation and amplified MYCN, with acquired resistance to the ALK inhibitor TAE684, through continuous exposure of increasing concentrations of the compound over time. We found that resistance was associated with downregulation of ALK activity, thus ruling out secondary mutations as a mechanism of resistance, and interestingly the other major oncogenic driver in neuroblastoma, MYCN, was downregulated as well. A comparative genome-wide expression analysis revealed global transcriptional downregulation in the resistant cells, as compared with their parental sensitive counterparts, in accordance with the aforementioned MYCN downregulation, as MYCN is known to act as a transcriptional amplifier in MYCN-amplified NB cells. Additionally, these resistant cells exhibited a 10-fold increased sensitivity to the BRD4 inhibitor JQ1, compared to sensitive cells, and MYCN as well as global gene expression were rescued after treatment. These results strongly suggest that resistance to ALK inhibition in ALKF1174L MYCN-amplified NB cells leads to a MYCN-independent enhanced BRD4 dependence, therefore providing a rationale for the use of BET inhibitors to overcome such resistance. Studies are currently underway to characterize the unique epigenetic landscape driving the resistant cells9 survival and therefore identify novel targets that could be taken advantage of in future therapeutic strategies. Citation Format: David Debruyne, Bandana Sharma, Nathanael Gray, Rani George. Resistance to ALK inhibition is associated with enhanced BRD4 dependence in ALK-mutated MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C24.

Abstract PR05: Targeting of CDK7 inhibits super-enhancer-associated oncogenic programs in MYCN-amplified tumor cells

The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a novel covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, would be useful therapy for cancers that are driven by MYC or its family members. Citation Format: Edmond Chipumuro, Eugenio Marco, Camilla L. Christensen, Nicholas Kwiatkowski, Tinghu Zhang, Clark M. Hatheway, Brian J. Abraham, Bandana Sharma, Caleb Yeung, Abigail Altabef, Antonio Perez-Atayde, Kwok-Kin Wong, Guo-Cheng Yuan, Nathanael S. Gray, Richard A. Young, Rani E. George. Targeting of CDK7 inhibits super-enhancer-associated oncogenic programs in MYCN-amplified tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR05.

Abstract LB-125: Selective inhibition of CDK7 targets MYCN-driven transcriptional amplification in neuroblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oncogenic MYC family transcription factors act as universal amplifiers of the existing gene expression program in many cancer cells, thus reducing rate-limiting constraints on growth and proliferation. Amplification of the MYCN gene defines approximately 50% of high risk neuroblastomas (NB), and is associated with aggressive disease and a poor clinical outcome. Here we exploit MYCN-driven global transcriptional amplification to specifically target MYCN-deregulated NB cells by inhibiting CDK7, a cyclin-dependent kinase with major roles in transcriptional initiation (as part of the TFIIH complex) and elongation (by activating CDK9/P-TEFb). For this purpose, we chose CDK7-IN-1, a newly developed, highly selective, first-in-class covalent inhibitor of CDK7, and then determined the effects of CDK7 inhibition on MYCN expression and global transcriptional activity. NB cells expressing high levels of MYCN were 10 times more sensitive to CDK7 inhibition than normal cells or NB cells not driven by amplified MYCN. CDK7-IN-1 was more active than its reversible (non-covalent) analogue and two pan-CDK inhibitors, roscovitine and flavopiridol. Cytotoxicity in treated MYCN -amplified NB cells resulted from G2 arrest and apoptosis. We observed a dose-dependent decrease in serine 2, 5 and 7 phosphorylation of RNA Pol II C-terminal domain only in MYCN -amplified NB cells, indicating that CDK7-IN-1 selectively inhibits RNA Pol II-mediated transcriptional initiation and elongation. Growth inhibition was accompanied by downregulation of MYCN and MYCN -associated transcriptional programs. CDK7-IN-1 significantly slowed tumor growth in a xenograft model of MYCN -amplified NB (median growth, 56.8% vs. 100% for vehicle-treated mice, P <0.05; n=6 per group) with tumors showing decreased MYCN expression. Mice remained free of toxicity over 4 weeks of CDK7-IN-1 treatment, suggesting that a therapeutic window may exist for NB cells with high MYCN expression. In conclusion, we show for the first time that selective suppression of MYCN expression and MYCN -associated transcriptional activity can be achieved through CDK7 inhibition, with associated antitumor effects in high-risk NB. Thus, CDK7 inhibition warrants further attention as a potential therapeutic strategy for MYCN-deregulated NB and perhaps other MYC-driven cancers. Citation Format: Edmond Chipumuro, Eugenio Marco, Tinghu Zhang, Camilla Christensen, Nicholas Kwiatkowski, Bandana Sharma, Clark Hatheway, Abigail Altabef, Brian J. Abraham, Kwok-Kin Wong, Guo-Cheng Yuan, Richard A. Young, Nathanael S. Gray, Rani E. George. Selective inhibition of CDK7 targets MYCN-driven transcriptional amplification in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2014-LB-125

Abstract 4454: The AXL tyrosine kinase receptor contributes to ALK-inhibitor resistance in neuroblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The crizotinib-resistant ALKF1174L mutation occurs de novo in neuroblastoma (NB) and as an acquired mechanism of resistance in ALK translocation-driven cancers, lending impetus to the development of novel ALK inhibitors with different modes of action. One such molecule is TAE684, a structurally unrelated ATP-competitive diaminopyrimidine derivative that has shown activity against ALKF1174L in both in vitro and in vivo models of ALK-driven cancers, such as lung cancer and NB. To identify mechanisms of acquired resistance to TAE684, we established in vitro models of resistance by serially exposing TAE684-sensitive SH-SY5Y NB cells (expressing ALKF1174L) to increasing concentrations of the compound over a prolonged period (> 1 year). The resulting TAE684-resistant SY5Y-TR cells exhibited >10-fold reduced sensitivity to TAE684 compared with parental cells. Three TAE684-resistant clonal lines (TR1-TR3) were analyzed, all of which showed downregulation of pALK. In addition, TR1 cells showed downregulation of pAKT but increased expression of pERK1/2 compared to parental cells, suggesting activation of compensatory signaling pathways. To identify candidate receptor tyrosine kinases (RTKs) that may account for this observation, we analyzed TR1 cells using phospho-RTK arrays. Upregulation of the AXL RTK was seen in the resistant cells as compared to the parental cells, and this was confirmed by immunoblotting in all the resistant clones. Concurrent with the role of AXL in tumor cell invasion, TR1 cells exhibited increased invasiveness in matrigel assays. Depletion of AXL expression by shRNA knockdown in TR1 cells led to their growth inhibition. TR1 cells were more sensitive to SKI-606, a Src/Abl inhibitor with activity against AXL, than parental cells. However, exposure to an Hsp90 inhibitor led to significant cytotoxicity in all the clones (IC50s<30nM), as compared to parental cells (IC50=304nM). Inhibiting Hsp90 in the TR1 cells led to a decrease in pAXL and pERK1/2 levels with a concomitant decrease in the binding of AXL to Hsp90. Analysis of AXL in TR1 cells ruled out mutation, genomic amplification or promoter demethylation as a basis for its increased expression; however, the AXL ligand, GAS6, was increased in the resistant clones. These studies demonstrate that AXL activation contributes to TAE684 resistance in ALKF1174L-expressing cells, and suggests that the same would be true for derivatives of TAE684 in clinical development. The finding that AXL is also involved in EGFR-targeted therapy in lung cancer suggests that AXL activation could be a general mechanism of resistance to tyrosine kinase inhibitor therapy. Citation Format: Namrata Bhatnagar, William Luther, Bandana Sharma, Qingsong Liu, Nathanael Gray, Rani E. George. The AXL tyrosine kinase receptor contributes to ALK-inhibitor resistance in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4454. doi:10.1158/1538-7445.AM2013-4454