Prolonged Disease Stabilization and Tolerability in a Nuclear Protein in Testis Midline Carcinoma Patient Treated with Dual Histone Deacetylase and Phosphoinositide 3-Kinase Inhibitor CUDC-907

Introduction: Nuclear protein in testis midline carcinoma (NMC) is a rare and extremely aggressive carcinoma (median survival < 7 months) with no effective treatment options. CUDC-907 is a novel small molecule inhibitor of histone deacetylase (HDAC) and phosphoinositide 3-kinase (PI3K) enzymes currently being investigated in multiple tumor types, including NMC. Case Report: A 61-year old female NMC patient enrolled on study CUDC-907-102 (NCT02307240) after rapidly progressing through two prior treatments. The patient’s assessable sites of disease consisted of right pleural effusion, right hilar soft tissue, and segment IV liver. Treatment was well tolerated with toxicities primarily consisting of manageable diarrhea and thrombocytopenia. The patient remains on active treatment after more than 32 months of stable disease. Discussion: Dysregulation of MYC in NMC is believed to play a central role in pathogenesis. CUDC-907 has demonstrated potent suppression of MYC expression and anti-tumor activity in preclinical NMC models, providing a mechanistic rationale for the prolonged disease stabilization observed here. The treatment of additional NMC patients with CUDC-907 is needed to further evaluate this promising report. Conclusion: This case demonstrates a rare success in the treatment of a devastating disease using only a novel small molecule, warranting further investigation of CUDC-907 in NMC.


Introduction
Nuclear protein in testis (NUT) midline carcinoma (NMC) is a genetically defined, rarely diagnosed, and poorly differentiated type of squamous cell carcinoma [1] [2] [3] [4]. Initially described in children and young adults, NMC has since been reported in patients of all age groups. Due to limitations on comprehensive cytogenetic and molecular assessments of adult carcinomas, accurate estimates on the actual prevalence and incidence of NMC are unavailable, and the current estimated numbers are likely to be highly underreported [1]. With the introduction of a diagnostic immunohistochemistry assay in 2009 and the establishment of an international NMC registry (http://www.nmcregistry.org/) in 2010, more patients will present with diagnosed NMC [1] [5]. The disease is extremely aggressive and invariably fatal despite aggressive chemotherapy and radiation treatments. With a reported median overall survival (OS) of less than 7 months, NMC represents a rapidly deadly disease with an urgent need for new effective treatment options [1] [3] [6].
The pathogenesis of NMC is genetically defined by the rearrangement and translocation of NUT most often with either the bromodomain-containing protein 4 (BRD4; [t(15;19) (q14;p13.1)]) or BRD3 (t(15;9) (q14;p34.2) ). The function of NUT is poorly understood and its expression is usually restricted to the germ cells of the testis and ovaries [5]. BRD4 and BRD3 belong to the bromodomain and extra-terminal domain family (BET) of proteins which bind to acetylated lysine residues on histone proteins and function as epigenetic readers and regulators of transcriptional activity [7]. Both proteins are believed to be essential in the transcription of oncogenes such as c-MYC, MYCN, BCL2, and FOSL1 [8]. In approximately 70% of cases, NMC is caused by the fusion of NUT (chromosome 15) with BRD4 (chromosome 19), forming the BRD4-NUT oncogene [9]. In the remaining cases, NUT is fused with BRD3 (25%) or to unidentified genes (5%). In recent reports, a NSD3-(nuclear receptor binding SET domain 3) NUT fusion was also identified, and a novel three-way translocation involving chromosomes 9, 15, and 19 [10] [11] [12]. The BRD-NUT fusion proteins appear to contribute to carcinogenesis by interacting with chromatin, triggering aberrant histone acetylation, and blocking cellular differentiation. In vitro studies have demonstrated that MYC is a key downstream target of BRD4-NUT, which associates with the MYC promoter and is required for MYC expression. The dysregulation of MYC by BRD-NUT fusion proteins is believed to play a central role in the pathogenesis of NMC [13] [14].
CUDC-907 is a novel oral small molecule that potently inhibits class 1 phosphoinositide 3-kinase (PI3K; α, β, and δ isoforms) as well as histone deacetylase (HDAC; class I and II) enzymes, blocking cellular proliferation and inducing apoptosis in a variety of cancer cell lines. Preclinical experiments have demonstrated that CUDC-907 dramatically decreased MYC mRNA and protein levels in multiple MYC-dependent cell and animal models. Furthermore, CUDC-907 has been shown to effectively inhibit the growth of human NMC cell lines in

Case Report
In January 2015 the patient, who is a never smoker, reported an approximate The patient has not required any thoracentesis for pleural effusion. The best overall response for every disease assessment by CT scan to date has been a non-complete remission (CR)/non-progressive disease (PD) ( Table 1).
As of 07 July 2018, the patient remains on active treatment for over 2 years in Cycle 42.

Discussion
Overall CUDC-907 treatment has been well tolerated with adverse events consisting primarily of mild-moderate diarrhea and intermittent thrombocytopenia.
Both of these events are commonly associated with CUDC-907 treatment as well as with other HDAC and PI3K inhibitors [16] [17]. Diarrhea events were generally managed by the use of loperamide or lomotil and budesonide. The Grade 1 colitis event reported in Cycle 12 was considered related to the presence of additional work up due to the patient's daughter also reporting events of diarrhea and may be associated with environmental factors. Furthermore, the patient also has a remote history of colitis which may have been related to prior exposure to Campylobacter jejuni. Thrombocytopenia events were managed by the use of dose holds and/or reductions.
The patient has reported encouragingly prolonged and ongoing disease stabilization for over 32 months. Due to the patient not having target lesions to measure, the direct quantitative impact of CUDC-907 on anti-tumor activity cannot be ascertained in this case. However, by the end of Cycles 10 and 26, the patient's segment IV liver and right hilar soft tissue lesions were absent, respectively (Table 1). In addition, thoracentesis has not been required on study, suggesting prolonged stability of the patient's pleural effusion since treatment was initiated. Hence, the patient's assessable sites of disease either gradually receded or remained stable through CUDC-907 treatment, demonstrating a marked improvement in disease control compared to their previous 2 therapies. Importantly the patient has demonstrated signs of improved quality of life since beginning treatment, such as the ability to exercise again. The median survival time for NMC patients from diagnosis is reported to be less than 7 months, and there is a greater than 80% likelihood of death within the first year of diagnosis for adult patients [1] [18] [19]. So far as current literature suggests, there appears only two cases of NMC patients reporting longer survival times than the patient presented here. In one case, a 16-year old boy with an endobronchial lesion (NUT variant) underwent resection and 4 cycles of adjuvant cisplatin and docetaxel followed by radiation therapy, was reported to be disease free 34 months after diagnosis [20]. In the other case, a 10-year old boy with a tumor in the iliac bone (BRD4-NUT positive) underwent combined modality therapy used for inoperable Ewing sarcoma, consisting of two courses of vincristine, doxorubicin, and ifosfamide, alternating with one course of cisplatin, doxorubicin, and ifosfamide for 35 weeks. During the first 2 cycles the patient also underwent hyper-fractionated accelerated radiotherapy to the tumor area. A post-treatment biopsy revealed no viable tumor cells and the patient was reportedly in continued complete remission 13 years later [21].
Presently there is no standard of care treatment in NMC due to the rarity and fulminant nature of the disease [18]. BET inhibitors have been shown to competitively inhibit BRD-NUT chromatin interactions, down regulate MYC and MYC-dependent gene expression, and promote cellular differentiation [22]. Objective responses in NMC have been reported from treatment with BET inhibitors OTX015/MK-8628 and GSK525762 [23] [24]. However, the effectiveness of this drug class is unclear due to a lack of clinical data and restored MYC expression through WNT signaling has been proposed as a mechanism for acquired resistance to BET inhibitors [24] [25] [26]. BET inhibition was unfortunately not a successful approach in this patient who progressed less than 3 months after treatment initiation. Another potentially promising approach in NMC has been the investigation of HDAC inhibitors (HDACi). NMC is hypothesized to be driven by an imbalance of histone acetylation/deacetylation leading to the arrest of differentiation and dysregulation of oncogenic MYC activity. Blocking endogenous histone deacetylation is believed to restore this imbalance and increase the expression of pro-differentiation genes [27]. Objective responses from the use of HDACi vorinostat have been reported in at least 2 NMC cases, demonstrating the potential effectiveness of such treatments [28] [29].
CUDC-907 potently inhibits HDAC and PI3K activities, both of which have been shown to individually suppress MYC-induced oncogenic transcriptional programs via different mechanisms. HDAC inhibition potently suppresses expression of MYC at the transcriptional level, while PI3K inhibition results in enhanced ubiquitin-mediated MYC protein degradation at the post-translational level. When combined, there is preclinical evidence that dual HDAC and PI3K inhibition results in synergistic suppression of MYC and anti-cancer effects in multiple MYC-driven models, including BRD4-NUT NMC [15] [30] [31] [32] [33]. CUDC-907 has also demonstrated promising and durable clinical activity in MYC-altered diffuse large B-cell lymphoma (DLBCL) patients [16]. This case highlights the general tolerability and promising activity of CUDC-907 in a tumor type that is rapidly fatal and which has few therapeutic options available. The study CUDC-907-102 continues to explore whether CUDC-907 may be an effective treatment option for more patients with this devastating disease.

Conclusion
NMC is a rare, but underdiagnosed, rapidly fatal disease for which there are no established effective treatment options available. The dysregulation of MYC is believed to be a central driver in the pathogenesis of NMC [13] [14]. CUDC-907, a dual HDAC and PI3K inhibitor, has demonstrated preclinical suppression of MYC expression and protein stability, potent anti-cancer activity in multiple MYC-driven tumor models including NMC, and promising clinical activity in MYC-altered DLBCL [15] [16]. In this case, a 61-year old female NMC patient who had previously progressed rapidly through treatment with platinum-based chemoradiotherapy and an investigational BET inhibitor has reported prolonged disease stabilization (ongoing at over 32 months) and manageable toxicities from treatment with CUDC-907. This case demonstrates a rare success in the treatment of a particularly devastating disease using only a novel oral small molecule inhibitor, warranting further investigation.