Suppl. Table 1. Binding mode of Debio 0617B to ABL. Suppl. Table 2. Physico-chemical and early ADME profile of Debio 0617B. Suppl. Table 3. Debio 0617B activity on a panel of 21 cancer cell lines. Suppl. Table 4. PK/PD Profile of Debio 0617B in mice with A549 tumors.

Legend for Supplementary Figures:1A,1B,1C,1D 2A,2B,2C;3A,3B

Legend for Supplementary Tables:1,2,3,4

•ACC tumours with diffuse NOTCH1 intracellular domain (NICD1) immunohistochemistry staining were associated with adverse clinical outcomes.•Multi-dimensional scaling analysis of RNA data showed that ACC with and without NICD1 diffuse staining tended to cluster separately.•ACC without NICD1 diffuse staining showed greater heterogeneity and those with SPEN or TP53 mutations clustered with NICD1 positive tumours.•Applying our gene expression data to a second dataset separated ACC into the ACC-I and II subclasses but correlation with NICD1 status was partial.

Tables S1-S4 show patient characteristics, kinome scan for Debio 0617B, selectivitly profile, GI50 values and FLT3-ITD inhibition capacity of Debio 0617B.

Figure S9 shows that Debio 0617B reduces spleen weight and cellularity in human PDX AML mice.

Figure S7 shows the CFU and re-plating capacity of CD34-positive stem/progenitor cells from healthy donors and newly diagnosed AML patients after exposure to Debio 0617B

Figure S5 shows the anti-leukemic activity of Debio 0617B in a MOLM-13-luciferase disseminated AML mouse model.

Figure S4 shows the pharmacokinetics/pharmacodynamics (PK/PD) of Debio 0617B in the rat MV-4-11 AML model.

Figure S3 shows response of MOLM-13 cells to Debio 0617B after silencing of STAT3 and STAT5.

Supplementary methods and materials shows supplemental procedures.

Figure S2 shows Debio 0617B-induced apoptosis of the FLT3-ITD-driven AML cell line MOLM-13.

Figure S1 shows expression of nuclear pSTAT3 and pSTAT5 in BM of newly diagnosed AML patients.

Figure S6 shows that the body weight of naive mice is not affected by Debio 0617B.

Suppl. Fig. 1 Characterization for pSTAT3 activation in cancer cell lines responding to Debio 0617B in vitro by Western blot analysis.Suppl. Fig. 2 A-C. Efficacy studies with Debio 0617B in A549, A2058 and A431 models: body weight loss/gain (%). Suppl. Fig. 3A. Histopathology of primary tumors in the 4T1 mammary tumor model. 3B. Efficacy of Debio 0617B in the 4T1 neoadjuvant mammary tumor model: body weight loss/gain (%).

Somatic mutations leading to activation of Notch signalling are recurrent in different blood cancers, including Tcell acute lymphoblastic leukaemia (TALL), chronic lymphocytic leukaemia (CLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL) and diffuse large Bcell lymphoma (DLBCL). 4 In mature Bcell lymphomas, the mutations most commonly occur in NOTCH1 and NOTCH2 genes and less frequently in genes coding for negative regulators of the pathway (such as DTX1, SPEN, FBXW7, MAML2). The presence of these mutations identifies subgroups of patients with similar clinical and biologic features. The genetically defined DLBCL subtype BN1 contains mostly activated Bcelllike (ABC) cases, and it is characterized by NOTCH1 mutations and active Notch signalling. NOTCH2 mutations, alongside BCL6 translocations, are characteristic of the BN2/C5 subtype, containing both ABC and germinal center Bcelllike (GCB) DLBCL, although no active Notch signalling has been observed in DLBCL cases belonging to this subtype. NOTCH2 mutations and NOTCH2 activation are common in splenic MZL. The hope of offering personalized therapies to these patients is sustained by the availability of drugs targeting the Notch signalling. One approach is the use of γsecretase inhibitors (GSI), small molecules that block the cleavage by an aspartyl protease of the NOTCH1– NOTCH4 receptors. The cleavage is fundamental to move the Notch intracellular domain (NICD) from the membrane to the nucleus, where it recruits transcriptional cofactors, such as RBPJ, and acts as transcriptional factor. In clinics, this class of agents has shown antitumour activity. However, diarrhoea, due to a skewed differentiation of intestinal stem cells towards goblet cells (goblet cell metaplasia), often represents a doselimiting toxicity. CB103 is a firstinclass, orally active, panNotch inhibitor that interferes with the RBPJ– NICD transcription complex. CB103 has shown in vitro and in vivo antitumour activity in TALL. Different to what is seen with GSIs, and due to its specific mechanism of action, there is preclinical and clinical evidence that CB103 does not induce goblet cell metaplasia. CB103 is currently in phase II (NCT03422679), based on the absence of the typical toxicities associated with GSI among the patients (41 with adenoid cystic carcinoma, 16 with colorectal cancer, four with breast cancer and two with prostate cancer) enrolled in the phase I study. So far, the clinical activity is represented by the achievement of longlasting (>6 months) stable disease in multiple patients with adenoid cystic carcinoma harbouring activating NOTCH alterations and a complete response in relapse/refractory TALL patient in combination therapy. Here, we have studied CB103 for its antitumour activity in 59 established human lymphoma cell lines, one canine and two murine lymphoma cell lines (detailed methods are presented in the Supplementary Materials). After 72 h of exposure, CB103 determined a dosedependent response (Figure 1; Table S1) and 14 cell lines had an IC50 below 10 μM: 5/20 (25%) GCB DLBCL, 3/7 (43%) ABC DLBCL, 3/9 (33%) MCL, 1/3 (33%) MZL, the canine DLBCL cell line CLBCL1 and the precursor TALL cell line Jurkat (Table S1). The range of active concentrations was in line with what is reported in TALL models. CB103 sensitive cell lines were then treated with another Notch inhibitor that had undergone early clinical development, the GSI crenigacestat (LY3039478). CB103 resulted overall superior in terms of antiproliferative activity and cell death (Figures S1 and S2). CB103 induced cell death by apoptosis in over one third of the sensitive cell lines (Figure S3). For almost all the cell lines, we can see a strong caspase activation at 72 h with increasing CB103 concentrations (500 nM, 2 μM, 8 μM or 15 μM). For some sensitive cell lines (Jurkat, HC1 and DOHH2) at some concentrations, we saw a lower caspase activation due to higher cell death occurred before the 72 h endpoint. Data were confirmed with cell cycle experiments, showing subG0 cell accumulation after CB103 treatment. Table S2 summarizes the genetic features of the sensitive cell lines. The Jurkat cell line, the most sensitive cell line and the only one with an IC50 below 1 μM, has a NOTCH1 activating mutation (c.4880G > A) (Figure S4). Another two sensitive cell lines (REC1 and RI1) have gainoffunction variants on NOTCH1 and NOTCH2, respectively, affecting the PEST domain and known to sustain Notch signalling. The other sensitive cell lines had mutations in genes involved in Notch pathway or mutated in the Notchdriven DLBCL clusters, but the contribution of the Notchrelated mutations in the sensitivity cannot be clearly determined. Expression levels of two Notch targets, HES1 and DTX1, did not differ between sensitive and resistant cell lines (Figure 1). Notch transcriptional network is strictly linked with that regulated by MYC, and activation of MYC was present in 57% (8/14) of sensitive cell lines (Figure S4). Of note, although two sensitive cell lines (DOHH2, OCILy1) Received: 25 August 2022 | Accepted: 15 November 2022

Notch is a key oncogenic pathway in several human cancers and to date, no targeted treatment of Notch activated cancers is available to patients. Therapeutic targeting of Notch has been an unresolved challenge due to severe on-target dose limiting toxicities associated with pan-Notch inhibition by either γ-secretase inhibitors or receptor/ligand targeting MAbs. At Cellestia Biotech, we have identified novel series of small molecule inhibitors of the Notch transcription complex. These molecules act as pan-Notch inhibitors and do not cause toxicities commonly associated with first- and second-generation Notch inhibitors currently tested in the clinic, thus providing a novel and unique opportunity to address a high unmet medical need. Our lead molecule, CB-103 is currently being investigated in Phase-1 dose escalation in cancer patients. Cellestia Biothech is further expanding its medicinal chemistry activities advancing the development of novel molecules for targeting transcription factors in cancer as well as non-cancer indications.

BACKGROUND:Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of multiple sclerosis (MS), a neuroinflammatory and demyelinating disease characterized by multifocal perivascular infiltrates of immune cells. Although EAE is predominantly considered a T helper 1-driven autoimmune disease, mounting evidence suggests that activated dendritic cells (DC), which are the bridge between innate and adaptive immunity, also contribute to its pathogenesis. Sirtuin 6 (SIRT6), a NAD+-dependent deacetylase involved in genome maintenance and in metabolic homeostasis, regulates DC activation, and its pharmacological inhibition could, therefore, play a role in EAE development.METHODS:EAE was induced in female C57bl/6 mice by MOG35-55 injection. The effect of treatment with a small compound SIRT6 inhibitor, administered according to therapeutic and preventive protocols, was assessed by evaluating the clinical EAE score. SIRT6 inhibition was confirmed by Western blot analysis by assessing the acetylation of histone 3 lysine 9, a known SIRT6 substrate. The expression of DC activation and migration markers was evaluated by FACS in mouse lymph nodes. In addition, the expression of inflammatory and anti-inflammatory cytokines in the spinal cord were assessed by qPCR. T cell infiltration in spinal cords was evaluated by immunofluorescence imaging. The effect of Sirt6 inhibition on the migration of resting and activated bone marrow-derived dendritic cells was investigated in in vitro chemotaxis assays.RESULTS:Preventive pharmacological Sirt6 inhibition effectively delayed EAE disease onset through a novel regulatory mechanism, i.e., by reducing the representation of CXCR4-positive and of CXCR4/CCR7-double-positive DC in lymph nodes. The delay in EAE onset correlated with the early downregulation in the expression of CD40 on activated lymph node DC, with increased level of the anti-inflammatory cytokine IL-10, and with a reduced encephalitogenic T cell infiltration in the central nervous system. Consistent with the in vivo data, in vitro pharmacological Sirt6 inhibition in LPS-stimulated, bone marrow-derived DC reduced CCL19/CCL21- and SDF-1-induced DC migration.CONCLUSIONS:Our findings indicate the ability of Sirt6 inhibition to impair DC migration, to downregulate pathogenic T cell inflammatory responses and to delay EAE onset. Therefore, Sirt6 might represent a valuable target for developing novel therapeutic agents for the treatment of early stages of MS, or of other autoimmune disorders.

NOTCH signalling is a key development pathway whose aberrant activation plays an onco-driver role in human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such as over expression of ligands/receptors, GOF mutations in NOTCH receptors, chromosomal translocations, or loss-of-function mutations in the negative regulators of the pathway. Activation of NOTCH due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). In addition to their limitations in targeting tumors harbouring chromosomal translocations in NOTCH receptors, the use of specific blocking antibodies against NOTCH receptors/ligands allows targeting of only a narrow spectrum of NOTCH positive tumors. On the other hand, GSIs act as pan-NOTCH inhibitors and thereby are able to target human tumors positive for NOTCH1-NOTCH4 receptors. However, clinical development of these pan-NOTCH inhibitors is hindered due to dose limiting toxicities (DLTs) associated with GSIs. The main DLTs associated with GSIs have been diarrhoea and vomiting due induction of goblet cell metaplasia in intestine, where NOTCH is known to regulate proliferation and differentiation of intestinal stem cells. A sustained blockage of NOTCH signalling leads to an inhibition of stem cell proliferation and differentiation of stem cells into mucous producing goblet cells. The effect on stem cell proliferation and differentiation is mediated via a subset of specific NOTCH target genes. Effect on proliferation is mostly driven by downstream target genes such as HES1, 3, and 5, cMYC and BMI1. The effect on stem cell differentiation is predominantly mediated by repression of MATH1. Therefore, MATH1 upregulation upon NOTCH inhibition is a pre-requisite to induce goblet cell metaplasia. Given the role of NOTCH signalling in human cancers and DLTs associated with 1st and 2nd generation NOTCH targeting agents (GSIs and blocking Abs), there is a need to develop novel pan-NOTCH inhibitors able to circumvent these DLTs. We have previously reported discovery and development of a novel class of pan-NOTCH inhibitors that blocks signalling by directly targeting the NOTCH transcription complex. Here we present further in vivo characterization of the development candidate CB-103. A comprehensive in vitro study demonstrated that CB-103 acts as a pan-NOTCH inhibitor. Furthermore, in vitro and in vivo pharmacological studies show that CB-103 circumvents DLTs, namely goblet cell metaplasia, associated with GSIs, due to the novel mode of action. We present molecular evidence that due to differential regulation of downstream target genes, CB-103 fully engages the NOTCH pathway in intestine and spares MATH1 gene repression and thereby does not cause goblet cell metaplasia. Citation Format: Rajwinder Lehal, Jelena Zaric, Michele Vigolo, Charlotte Urech, Maximilien Murone, Freddy Radtke. Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2695.

Abstract NOTCH signalling is a key development pathway whose aberrant activation is known to play a role in multiple human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such over expression of ligands/receptors, GOF mutations in NOTCH receptors, including protein stabilizing mutations in the PEST domain of NOTCH, chromosomal translocations, or loss-of-function mutations in the E3 ubiquitin ligase FBXW7 and other negative regulators of the pathway (SPEN, NUMB). Activation of signalling due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). However, in human tumors where NOTCH signalling is constitutively activated due to chromosomal translocations in the NOTCH receptors (NOTCH1 and 2), none of the above-mentioned strategies will be effective. Moreover, due to on-target and off-target toxicities associated with blocking antibodies and GSIs, these anti-NOTCH agents failed to advance in clinical trials, although some of them showed signs of clinical efficacy. Given the role of NOTCH signalling in human tumors, there is a need to identify novel targets in the NOTCH pathway and develop new and more selective anti-NOTCH agents. To inhibit pan-NOTCH signalling in human tumors independently of the mechanisms of NOTCH activation, and in the most downstream part of the pathway, we have previously reported the discovery of a new class of small molecules able to target the NOTCH transcription complex enabling the specific inhibition of NOTCH target gene expression (e.g. cMYC, HES1, DTX1, CCND1). These small molecules act as protein-protein interaction inhibitors, and thereby compromise the assembly of functional NOTCH transcription complex. Here we present further in vitro and in vivo characterization of the lead molecule CB-103. The anti-cancer activity of CB-103 was extensively profiled in several human cancer cell lines representing NOTCH positive solid tumors, leukemias and lymphomas. Moreover, CB-103 responsiveness of these cell lines correlates with a downregulation of the NOTCH signature following treatment with CB-103. Specifically, we will present data outlining the in vivo pharmacokinetic and pharmacodynamic properties of CB-103. Citation Format: Rajwinder Lehal, Charlotte Urech, Michele Vigolo, Maximilien Murone, Freddy Radtke. Characterization and profiling of CB-103, a novel small-molecule protein-protein interaction inhibitor targeting the NOTCH transcription complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5799.