WO2020008046A1

WO2020008046A1 - Biomarker

Info

Publication number: WO2020008046A1
Application number: PCT/EP2019/068134
Authority: WO
Inventors: Bassam Claude JANJI; Muhammad Zaeem NOMAN; Guy Joseph BERCHEM
Original assignee: Sprint Bioscience Ab
Priority date: 2018-07-06
Filing date: 2019-07-05
Publication date: 2020-01-09

Abstract

An in vitro method of determining the in vivo immunostimulating effect resulting from pharmacological inhibition of Vps34 by a Vps34 inhibitor is disclosed, comprising: in vitro analysing a test sample originating from a source to determine a level of at least one biomarker, wherein the at least one biomarker is selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFN_γ, and preferably selected from CCL5 and CXCL10, wherein an elevated level of said at least one biomarker in said test sample, relative to a reference level, indicates immunostimulation in said source.

Description

BIOMARKER

FIELD OF THE INVENTION

The present invention relates to biomarkers of inhibition of autophagy or related processes involving Vps34, and to methods of use of such

biomarkers.

BACKGROUND

Cancer immunotherapy is a promising and groundbreaking treatment for many cancers. Bursting the activity of the patients’ own innate immune system by different approaches is emerging as a major therapeutic possibility for cancer treatment. The existence of immune“cold” tumours, characterized by the lack of infiltrating immune cells, or presence of inactive immune cells, supports the therapeutic rationale of inducing an inflammatory response in the tumour that would increase recruitment of immune cells and hence facilitating tumour killing by the immune system. However, immune cells present in the tumour can encounter an immunosuppressive environment, as a

consequence of either the activity of suppressing immune cells or activation of checkpoint inhibitors. The latter exist in several immune cell types, such as NK-, T- and dendritic cells, macrophages and cancer cells.

One mediator of an inflammatory response is STING (Stimulator of Interferon Genes). STING is activated by cytosolic DNA and induces an increase in the secretion of cytokines and an upregulation of inflammatory signalling that attracts immune cells to tumours. Agonists of STING are therefore being tested as anti-cancer treatment. Another anti-cancer immunotherapy of interest is immune checkpoint blockade, including, for instance, antibodies targeting programmed death 1 (PD-1 ) or programmed death ligand 1 (PD-L1 ). However, satisfactory clinical responses to immune checkpoint blockade are only observed in a limited number of cancer patients and tumour types. The majority of patients treated with immune checkpoint inhibitors only reap short- term benefits or no benefits at all [Smyth MJ, Ngiow SF, Ribas A, & Teng MW (2016) Nat Rev Clin Oncol 13(3):143-158]. Therefore, there is a strong clinical need to improve immune checkpoint-based therapies.

Durable clinical responses using anti-PD-1/PD-L1 -based therapy have been associated with T cell-inflamed tumour microenvironment favoring the infiltration of functional cytotoxic T lymphocytes (CTL) [Tang H, et al. (2016) Cancer Cell 29(3):285-296; Tang H, Wang Y, Chlewicki LK, Zhang Y, & Fu Y- X (2016) The Journal of Immunology 196(1 Supplement):74.72] Significant efforts to identify therapeutic approaches that enhance immune cell infiltration into the tumour bed are currently undertaken [Tumeh PC, et al. (2014) Nature 515(7528):568-571] It has been reported that melanoma tumours containing a T cell infiltrate expressed high level of chemokines CXCL9 and CXCL10 [Monteagudo C, Martin JM, Jorda E, & Llombart-Bosch A (2007) J Clin Pathol 60(6):596-599]; however the putative mechanism(s) underlying the

expression of these chemokines remains largely unknown.

Autophagy is a cellular process involved in the degradation and recycling of cytoplasmic contents in well-defined structures called autophagosomes. The fusion of autophagosomes with lysosomes leads to the degradation of sequestered materials by lysosomal hydrolases. Several reports have suggested that autophagy inhibition can enhance the antitumour efficacy of chemotherapy or targeted therapies.

It has been shown that autophagy activation in tumour cells plays a major role in impairing the anti-tumour immune response [Janji B, et al. (2016)

Oncotarget 7(14):17591 -17607; Noman MZ, Janji B, Berchem G, Mami- Chouaib F, & Chouaib S (2012) Autophagy 8(4):704-706; Viry E, et al. (2014) Autophagy 10(1 ):173-175; Viry E, et al. (2016) Front Oncol 6:246] and its targeting inhibits the tumour growth and improves CTL and Natural Killer (NK)-mediated killing [Baginska J, et al. (2013) Proc Natl Acad Sci U S A 110(43):17450-17455 ; Messai Y, et al. (2014) Cancer Res 74(23):6820- 6832; Noman MZ, et al. (2011 ) Cancer Res 71 (18):5976-5986]. Interestingly, autophagy has been found to have a key role in the degradation of damaged nuclear DNA in cells deficient of Dnase2a. It has been shown that DNA accumulates in autophagy-deficient cells, resulting in STING-mediated inflammation [Barber GN (2015) Nat Rev Immunol 15(12): 760-770].

Furthermore, the STING protein itself has been shown to be degraded by autophagy, resulting in attenuated STING signaling [Prabakaran et al (2018) EMBO J. 2018 Apr 13;37(8)]. More recently, it has also been shown that targeting autophagy related gene Beclin-1 inhibited melanoma tumour growth by inducing the infiltration of functional NK cells into the tumour

microenvironment by a mechanism involving the release of CCL5 by Beclin-1 - defective tumour cells [Mgrditchian T, et al. (2017) Proc Natl Acad Sci U S A 1 14(44):E9271 -E9279]. However, the impact of autophagy inhibition on the regulation of the antitumour immune response is still only partially

understood.

The phosphoinositide 3-kinase (PI3K) family consists of three classes: class I, II and III. The shared name arises from the fact that all of these enzymes use phosphoinositides as substrates for their kinase activity. Still, the biological changes arising from their respective enzymatic activities differ significantly. PI3Ks from these three different classes catalyze the production of three distinct phosphoinositides. In vivo, there is significant support for class I PI3K- mediated synthesis of Ptdlns(3,4,5)P3 (and indirectly, Ptdlns(3,4)P2, class III PI3K-mediated synthesis of Ptdlns(3)P, and to a lesser extent, class II PI3K- mediated synthesis of Ptdlns(3)P and Ptdlns(3,4)P2 [Jean and Kiger, J Cell Sci. 2014, 127(5), 923-928]. Their different subunit composition, including catalytic and regulatory subunits, contribute not only to their differential enzymatic activity, but also to the differences in signaling pathways regulated by the different PI3K.

The PI3K Class III, vacuolar protein sorting 34 (Vps34, PIK3C3) forms a heterodimer with its regulatory subunit p150 (Vps15) and Beclin-1 (Becnl ). Although the role of Vps34 has been mostly related to the regulation of autophagy, new data connects this complex, along with other ad-hoc regulators, to the regulation of other vesicular trafficking events such as endocytosis, exocytosis and micropinocytosis.

An existing method of assessing autophagy inhibition in cells e.g. following treatment with a Vps34 inhibitor involves detection of microtubule-associated protein 1 A/1 B-light chain 3 (LC3) tagged with green fluorescent protein

[Honda et al., ACS Med. Chem. Lett., 2016, 7 (1 ), pp 72-76]. However, a positive signal did not correlate with a reduction of tumour volume, and this method has been found be unpredictive in vivo.

There remains a need in the art for improved means of assessing the effect of inhibition of autophagy and/or thereto related processes, as well as for improved therapeutic strategies for treating cancer.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome this problem, and to provide methods useful in the development of and/or clinical use of

autophagy inhibitors, in particular Vps34 inhibitors, and therapeutic strategies involving cancer immunotherapy.

According to a first aspect of the invention, this and other objects are achieved by an in vitro method of determining the in vivo immunostimulating effect resulting from pharmacological inhibition of Vps34 by a Vps34 inhibitor, comprising: in vitro analysing a test sample originating from a source to determine a level of at least one biomarker, wherein the at least one biomarker is selected from the group consisting of CCL2, CCL3, CCL4,

CCL5, CXCL9, CXCL10 and IFNy, and preferably selected from CCL5 and CXCL10, wherein an elevated level of said at least one biomarker in said test sample, relative to a reference level, indicates immunostimulation in said source. The immunostimulation typically results at least in part from inhibition of Vps34 and/or its complex with Beclin-1 achieved by said Vps34 inhibitor. It is shown herein that inhibiting Vps34 makes cancer tumours eligible for, and thus improves the therapeutic benefit of, anti-PD-1/PD-L1

immunotherapy. Hence, cancer patients that would normally be non- responders to immune checkpoint blockade immunotherapy can still be successfully treated with a combination therapy, if an immunostimulating treatment with a Vps34 inhibitor is found to be effective. The present inventors have found that certain proteins (or their encoding genes) can be surprisingly useful as biomarkers for in vitro determining, evaluating and/or predicting the effect of an autophagy inhibitor, in particular a vps34 inhibitor, exerted in vivo. Thus, the method of the present invention provides a means to predict the effectiveness of a Vps34 inhibitor and may provide valuable information that can be used clinically to improve treatment of many cancer types.

Thus, the invention, according to the various aspects described herein, may contribute to increasing the effectiveness of immunotherapy treatments and/or provide a means to more accurately determine treatment progression and patient selection for improved therapeutic results.

As used herein, a“Vps34 inhibitor” (also referred to as“Vps34i”) refers to a compound capable of inhibiting enzymatic activity of vacuolar protein sorting 34 (Vps34). Inhibition of enzymatic activity may be demonstrated in a biochemical assay, for instance a method as described in publications WO2017140841 and WO2017140843 which determines an IC50 value. In the context of the present invention, a compound exhibiting an IC50 value of 100 nM or less may be considered to be a Vps34 inhibitor.

Thus, the Vps34 inhibitor typically acts by pharmacological inhibition, by inhibiting enzymatic activity of the Vps34 protein.

As used herein,“immunostimulating effect” is understood to mean the activation of an immune response, systemically and/or locally, such as in the vicinity of a cancer cell or tumour. An immunostimulating effect may be evidenced by local or systemic activation, increase in the number of and/or local infiltration of immune cells, such as NK cells or T cells, e.g. CD8⁺ T cells; and/or by systemic or local increase or release of inflammatory molecules, such as cytokines, e.g. interferon gamma (IFNy), or chemokines. In fact, among the biomarkers identified by the present inventors are found several cytokines and chemokines. An exampe of an immunostimulatory effect is an anticarcinogenic effect, such as leading to an induction of cancer regression.

The term "biomarker" generally refers to any biological compound, such as a protein and a fragment thereof, a peptide, a polypeptide, or other biological material whose presence, absence, level or activity is correlative of or predictive of a characteristic. A biomarker may be recognized, for example, by an antibody (or an antigen-binding fragment thereof) or other specific binding protein(s). Reference to a biomarker may also include its isoforms, preforms, mature forms, variants, degraded forms thereof, and metabolites thereof.

The term "determining" the amount or level of a biomarker as referred to herein refers to the quantification of the biomarker, e.g. to measuring the level of the biomarker in the sample, employing appropriate methods of detection. The terms "measuring" and "determining" are used herein interchangeably. Determining a level of the at least one biomarker may mean determining the mRNA expression level of said at least one biomarker, and/or determining the protein concentration of said at least one biomarker in said sample. A step of determining the amount or level of a biomarker may be carried out in vitro.

To be of greatest clinical use, a biomarker is preferably detectable in a sample that can be obtained from a living subject without substantial health risks, for instance a blood sample. Preferably, the biomarker is quantitatively detectable, using established analytical methods. Lastly, to be clinically useful the level of the biomarker in a sample preferably significantly correlates with an in vivo characteristic or clinical condition of interest. The above method may comprise i) providing at least one test sample; and ii) in vitro analysing said test sample to determine the level of said at least one biomarker, wherein an elevated level of said at least one biomarker, relative to a reference level, indicates immunostimulation.

In embodiments, the reference level, to which the biomarker level detected in the test sample is compared, may be based on a reference sample from a source that has not been exposed to or subjected to treatment with a Vps34 inhibitor. That is, the level of the at least one biomarker is determined in said reference sample, and this level may represent the reference level.

Preferably, the reference sample is obtained, prior to exposure of the Vps34 inhibitor, from the same source from which the test sample is obtained after exposure to or treatment with a Vps34 inhibitor.

The source from which the test sample, and optionally also a reference sample, originates may be an in vitro or ex vivo source, such as a cell, optionally a cancer cell, a cell culture, or an extracted tumour or other tissue maintained in vitro. The source may also be a living human or animal subject, from which a sample has been obtained prior to carrying out the method according to the first aspect above. Thus, the source may be a living source, such as a cell, a tissue, or a human or animal.

The method may comprise in vitro analysing said reference sample and said test sample to determine the level of at least one biomarker in each of said samples, wherein an elevated level of said at least one biomarker in the test sample relative to the level of the biomarker in the reference sample indicates immunostimulation in said source.

In embodiments, the method may comprise

i-a) providing a biological reference sample originating from a living source that has not been subjected to treatment with a Vps34 inhibitor;

i-b) providing a biological test sample originating from a living source subjected to treatment with a Vps34 inhibitor; ii) in vitro analysing said biological reference sample and said biological test sample to determine the level of at least one biomarker in each of said samples, and wherein an elevated level of said at least one biomarker in the biological test sample relative to the biological reference sample indicates immunostimulation in said source treated with a Vps34 inhibitor. Typically, the reference and test samples may originate from the same source, such as a cell culture, or a human or animal subject.

The test sample as used in the methods according to various aspects of the invention may comprise a material selected from the group consisting of: cell culture supernatant, tumour plasma, and bodily fluids such as blood, blood plasma and urine, and preferably is blood plasma. Similarly, where a reference level is based on a reference sample comprising a material selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine, and preferably is blood plasma. In particular, in embodiments the test sample may be blood plasma or tumour plasma, preferably blood plasma; and the reference sample may also be blood plasma or tumour plasma, preferably blood plasma.

In a second aspect, there is provided a method of determining and/or predicting the immunostimulating effect of treatment with a Vps34 inhibitor in a subject afflicted with cancer, wherein said treatment comprises

administration of a Vps34 inhibitor to said subject, said method comprising:

(i) determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample obtained from said subject, and

(ii) comparing the level of said biomarker in said test sample to a reference level,

wherein an increase of the level of said biomarker in the test sample relative to the reference level indicates or predicts an effective treatment with the Vps34 inhibitor. The information (i.e., whether or not a Vps34 inhibitor treatment is effective) gained from the methods according to either of the first and second aspects above is useful for deciding on further treatment(s) of a patient, in particular a cancer patient, and, as a more well-informed decision regarding the subsequent therapeutic strategy may be taken, the above method may increase the chances of a successful treatment of a patient, and/or avoid, or reduce the risk of, subjecting a patient to a further treatment to which he or she is likely to be non-responsive. As such, the information gained from the methods may be used as a basis for a decision to continue treatment with Vps34 inhibitor, either with a maintained dose, an increased dose or a decreased dose. Alternatively, the decision may be, especially after a time of increased or prolonged dosing, to stop treatment with Vps34 inhibitor.

The term "treatment" includes any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Hence, "treatment" includes prophylactic and therapeutic treatment.

The reference level may be based on a reference sample as described above, typically obtained from said subject prior to said treatment.

Furthermore, the test sample and optionally the reference sample may be as described above in connection with the first aspect.

In embodiments, the method may comprise the steps of

a) providing a reference sample originating form said subject prior to said treatment;

b) administering a Vps34 inhibitor to said subject;

c) providing a test sample originating from said subject;

d) determining the level of said at least one biomarker in each of said reference sample and said test sample; and

e) comparing the level of said at least one biomarker in said test sample to the level of said at least one biomarker in the reference sample, wherein an increased level of said at least one biomarker in said test sample indicates or predicts an effective treatment with the Vps34 inhibitor.

In embodiments, in step i) the mRNA expression level of said at least one biomarker is determined in said test sample and optionally in said reference sample.

In embodiments, in step i) the protein concentration of said at least one biomarker is determined in said test sample and optionally in said reference sample.

The test sample and optionally the reference sample may be as described above. In preferred embodiments, the test sample and the reference sample, if any, is or comprises blood plasma. In embodiments, the method may comprise determining the protein concentration of the at least one biomarker in, respectively, a reference sample of blood plasma and in a test sample of blood plasma, and comparing the determined levels.

In a third aspect, the invention provides a method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, the method comprising

i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor; and

ii) comparing the level of said biomarker in said test sample to a predetermined reference level,

wherein, if the level of said biomarker in said test sample is elevated relative to the reference level, selecting immunotherapy as a further treatment optionally in combination with treatment with the Vps34 inhibitor.

Suitable immunotherapies are exemplified below. Optionally, the step i) may further comprise determining the level of said at least one biomarker in a reference sample obtained from said subject prior to treatment with the Vps34 inhibitor, and the predetermined reference level may then correspond to the level of the biomarker in the reference sample.

In a fourth aspect, the invention provides a method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, the method comprising

i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample originating from said subject who has received treatment with a Vps34 inhibitor; and ii) comparing the level of said biomarker in said test sample to a predetermined reference level, and

iii), if the level of said biomarker in said test sample is elevated relative to the reference level, selecting continued treatment with the Vps34 inhibitor.

Optionally, if the level of said biomarker in said test sample is not elevated relative to the reference level, the subject may be selected for further treatment with the Vps34 inhibitor at an increased dose. As a further option, if the level of said biomarker in said test sample is not elevated relative to the reference level, further treatment with the Vps34 inhibitor may be

discontinued.

In a fifth aspect, there is provided a method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(c) determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in said test sample;

(d) comparing the level of said at least one biomarker determined in step iii) to a predetermined reference level; and (e) if the level of said at least one biomarker in the test sample is increased relative to said reference level, further subjecting said subject to treatment with immunotherapy.

Optionally, the subject may be subjected to further treatment with the Vps34 inhibitor simultaneously, intermittently or sequentially with the

immunotherapy.

Optionally, prior to the step of (a) administering a Vps34 inhibitor to said subject, the method may comprise obtaining a reference sample from said subject, and further comprising determining the level of said at least one biomarker in said reference sample, and wherein said predetermined reference level represents the level of said at least one biomarker determined in said reference sample.

In a further aspect, there is provided a method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(d) comparing the level of said at least one biomarker determined in step iii) to a predetermined reference level; and

(e) if the level of said at least one biomarker in the test sample is increased relative to said reference level, selecting continued treatment with the Vps34 inhibitor.

Optionally, if the level of said biomarker in said test sample is not elevated relative to the reference level, the subject may be selected for further treatment with the Vps34 inhibitor at an increased dose. As a further option, if the level of said biomarker in said test sample is not elevated relative to the reference level, further treatment with the Vps34 inhibitor may be discontinued.

Determining a level of the at least one biomarker may comprise determining the mRNA expression level of said at least one biomarker, and/or determining the protein concentration of said at least one biomarker in said sample is determined, as described herein.

The test sample and optionally the reference sample may be as described herein.

By“immunotherapy” is understood a therapy whose rationale is based on modification, manipulation or use of elements of the immune system including, for instance, engineered immune cells, antibodies or small molecules that block immune cell activity, antibodies or small molecules that stimulate immune cells activity to improve or restore the function of the immune system.

The immunotherapy may be an immune checkpoint blockade-based therapy. Immune checkpoint blockade is understood as the use of antibodies or small molecules that inhibit the interaction of checkpoint inhibitors, which are proteins expressed at the surface of several cell types, which, when linked to their respective interaction partner(s), suppress the activity of cells of the immune system. Non-limiting examples of immune checkpoint blockade therapy include anti-PD-1 and PD-L1 based immunotherapy and macrophage immune checkpoint immunotherapy, and may optionally comprise treatment with an agent selected from the group consisting of Nivolumab,

Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Tislelizumab, and Sintilimab or from the group consisting of an anti-CD47 antibody and an anti-SIRPalpha antibody. Alternatively or additionally the immunotherapy may comprise one or more of the following: an anti-CTLA-4 based immunotherapy, a CAR-T cell therapy, treatment with a CD3-targeted bispecific antibody, an anti-NKG2A based immunotherapy, an anti-KIR based immunotherapy, and STING based immunotherapy comprising treatment with a STING agonist.

In a further aspect, the invention provides a method of identifying a subject responsive to, or suitable to receive, treatment with a Vps34 inhibitor wherein the subject is afflicted with cancer, comprising

- providing a test sample originating from said subject;

- determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in said test sample; and

- comparing the level of said at least one biomarker in said test sample to a predetermined threshold value,

wherein, if the level of said at least one biomarker in said test sample is lower than said predetermined threshold value, said patient is identified as likely to be responsive to immunostimulating treatment using a Vps34 inhibitor.

Optionally, if the level of said at least one biomarker in said test sample is lower than said predetermined threshold value, the patient may be identified as suitable to receive a combination treatment using a Vps34 inhibitor and immunotherapy, such as an immune checkpoint blockade based

immunotherapy, or another type of immunotherapies as indicated above, including combinations of such immunotherapies.

In a further aspect, an in vitro method of determining the in vivo

immunostimulating effect resulting from pharmacological inhibition of Vps34 by a Vps34 inhibitor, comprising: in vitro analysing a test sample originating from a source to determine a level of at least one biomarker, wherein the at least one biomarker is selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy, and preferably selected from CCL5 and CXCL10, wherein an elevated level of said at least one biomarker in said test sample, relative to a reference level, indicates immunostimulation in said source. The immunostimulation typically results at least in part from inhibition of Vps34 and/or its complex with Beclin-1 achieved by said Vps34 inhibitor.

In a further aspect, there is provided a method of determining and/or predicting the immunostimulating effect of treatment with a Vps34 inhibitor in a subject afflicted with cancer, wherein said treatment comprises

administration of a Vps34 inhibitor to said subject, said method comprising:

(i) determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample obtained from said subject, and

wherein an increase of the level of said biomarker in the test sample relative to the reference level indicates or predicts an effective treatment with the Vps34 inhibitor.

In a further aspect, the invention provides a method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, the method comprising

i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample originating from said subject; and

ii) comparing the level of said biomarker in said test sample to a predetermined reference level, and

iii)if the level of said biomarker in said test sample is elevated relative to the reference level, selecting immunotherapy as a further treatment for said subject optionally in combination with treatment with the Vps34 inhibitor.

In a further aspect, the invention provides a method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, the method comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample originating from said subjectr; and

iii) if the level of said biomarker in said test sample is elevated relative to the reference level, selecting continued treatment with the Vps34 inhibitor.

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(c) determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in said test sample;

(e) if the level of said at least one biomarker in the test sample is increased relative to said reference level, further subjecting said subject to treatment with immunotherapy.

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(d) comparing the level of said at least one biomarker determined in step iii) to a predetermined reference level; and (e) if the level of said at least one biomarker in the test sample is increased relative to said reference level, selecting continued treatment with the Vps34 inhibitor.

- providing a test sample originating from said subject;

- determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in said test sample; and

In further aspects, the invention provides the use of at least one cytokine selected from the group consisting CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy as a biomarker of inhibition of Vps34 in a cancer cell. In particular, the biomarker may be CCL5 and/or CXCL10. Said use may be in vitro use. Optionally, at least two biomarkers selected from this group are detected.

In further aspects, the invention provides the use of at least one cytokine selected from the group consisting CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy as a biomarker of inhibition of Vps34 in a cancer cell. In particular, the biomarker may be CCL5 and/or CXCL10. Said use may be in vitro use. Optionally, at least two cytokinesare detected.

The invention also relates to a biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy for use in a method of treating cancer. The invention also relates to a biomarker selected from CCL2, CCL3, CCL4,

CCL5, CXCL9, CXCL10, CXCL1 1 and IFNy for use in a method of treating cancer. The method of treating cancer typically involves treatment with a Vps34 inhibitor, and in cases where the biomarker is detected at an increased level in relation to a reference, the treatment may involve a combination of treatment with a Vps34 inhibitor and immunotherapy. In cases where the biomarker is not detected at an increased level in relation to a reference, the further treatment may involve in increased dosing of the Vps34 inhibitor, optionally in combination with immunotherapy, or the further treatment may consist in a different treatment, such as radiotherapy or chemotherapy.

In embodiments, the invention provides a combination of biomarkers selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy for use in a method of treating cancer, in particular as biomarkers.

In embodiments, the invention provides a combination of biomarkers selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL1 1 and IFNy for use in a method of treating cancer, in particular as biomarkers.

In embodiments of any of the herein disclosed aspects of the invention, the at least one biomarker may be selected from CCL5 and CXCL10.

In any of the above-described aspects of the invention, the Vps34 inhibitor may be 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-phenyl-1 -piperidyl]-1 H- pyridin-2-one, generally referred to as“SB02122”, 4-[(3R)-3-methylmorpholin- 4-yl]-6-[2-(trifluoromethyl)phenyl]-1 FI-pyridin-2-one, generally referred to as SB02008, 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2S)-2-(trifluoromethyl)-1 - piperidyl]-1 H-pyridin-2-one, generally referred to as SB02877, 4-(1 H- pyrazolo[3,4-b]pyridin-4-yl)-6-[2-(trifluoromethyl)-1 -piperidyl]-1 FI-pyridin-2-one, generally referred to as SB03388, methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H- pyridin-4-yl]-2-pyridyl]carbamate, generally referred to as SB03706, 4-[(3R)-3- methylmorpholin-4-yl]-6-[4-pyrrolidin-1 -ylsulfonyl-2-(trifluoromethyl)piperazin- 1 -yl]-1 H-pyridin-2-one, generally referred to as SB03439, (2S)-1 -[(5-chloro-3- pyridyl)methyl]-8-[(3R)-3-methylnnorpholin-4-yl]-2-(trifluoronnethyl)-3,4- dihydro-2H-pyrimido[1 ,2-a]pyrimidin-6-one, generally referred to as “SAR405”, or 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)-1 - piperidyl]-1 H-pyridin-2-one, generally referred to as“SB02024”, and occasionally herein also as“SB2024” or“2024”.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in more detail, with reference to the appended drawings, in which:

Figure 1A-B are graphs showing the tumour growth curve of vehicle or Vps34 inhibitor (SB02024 and SAR405) treated B16-F10 (Fig. 1A) or CT26 (1 B) tumour-bearing mice (n=10, average of 3 independent experiments). Tumour burden was assessed by calliper measurements on days 9, 1 1 , 13, 15 and 17 after inoculation.

Figure 2 is a graph illustrating flow cytometry quantification of infiltrating CD45⁺ leukocytes (gated in live cells) in vehicle or Vps34 inhibitor (SB02024 or SAR405) treated B16-F10 (left) or CT26 (right) tumours on day 15.

Figure 3A-B are graphs showing quantification of the percent of different subpopulations of anti-tumour immune effector cells infiltrating vehicle or Vps34 inhibitor (SB02024 and SAR405) treated B16-F10 (Fig. 3A) or CT26 (3B) tumours. Indicated cells are NK cells (NK), CD8 T cells (CD8), CD4 effector T cells (CD4 eff), dendritic cells (DC), type 1 macrophages (M1 ).

Figure 4A-B are graphs showing quantification of the percent of PD1 ⁺ NK, CD8 and CD4 effector T cells (CD4 eff) in vehicle or Vps34 inhibitor

(SB02024 or SAR405) treated B16-F10 (Fig. 4A) or CT26 (4B) tumours.

Figures 5A-B are graphs showing quantification of the percent of PD-L1 ⁺ cells in CD45 tumour cells, CD1 1 b⁺ cells, F480⁺, dendritic cells (DC) and Myeloid derived suppressor cells (MDSC) in vehicle or Vps34 inhibitor (SB02024 or SAR405) treated B16-F10 (Fig. 5A) or CT26 (5B) tumours. In terms of quantifying the immune cells infiltrating the tumours, the defined

subpopulations were gated in live cells. Each dot represents one tumour in all of the panels. The data are reported as the average of 5 mice per group.

Figure 6 is a graph showing the growth curve of vehicle or Vps34 inhibitor (SB02024 and SAR405) treated B16-F10 tumours in isotype-treated (iso) or CD8-depleted (aCD8) C57BL/6 mice. The data are reported as the average of 5 mice per group.

Figures 7A-B are graphs illustrating the expression level of CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11 mRNA, in Vps34 inhibitor (SB02024, middle bar, and SAR405, right bar) treated B16-F10 (Fig. 7A) or CT26 (7B) cells reported as fold change (FC) compared with vehicle treated cells

(control, left bar).

Figures 8A-B are graphs illustrating quantification of the secreted CCL5 protein levels and CXCL10 protein levels in cell supernatants, of vehicle treated (control, left bar) or Vps34 inhibitor (SB02024, middle bar, or SAR405, right bar) treated B16-F10 (Fig. 8A) or CT26 (8B) cells by ELISA reported in pg/ml. The data are reported as the average of 3-6 independent experiments.

Figure 9 shows two graphs illustrating the expression level of CCL5 mRNA (left graph) and CXCL10 mRNA (right graph), in Vps34 inhibitor (SB02024 or SAR405) treated B16-F10 tumour lysates (right and middle bars, respectively; n=8) reported as FC, compared with the vehicle treated tumour lysates (left bar; n=8).

Figures 10A-B are graphs illustrating quantification of the secreted CCL5 and CXCL10 proteins levels in tumour plasma of B16-F10 (Fig. 10A) or CT26 (10B) tumours untreated (squares) or treated with Vps34 inhibitor (SB02024 or SAR405) (diamonds and circles, respectively; n=10) by ELISA reported in pg/ml standardized to excised tumour weight in grams (g).

Figures 11A-B are graphs illustrating quantification of the secreted IFNy proteins levels in tumour plasma of B16-F10 (Fig. 11A) or CT26 (11 B) tumours untreated (squares) or Vps34 inhibitor (SB02024 or SAR405) treated, (diamonds and circles, respectively, n=10) by ELISA reported in pg/ml standardized to excised tumour weight in grams (g).

Figures 12A-B are graphs illustrating quantification of the secreted CCL5 and CXCL10 protein levels in blood plasma of untreated or Vps34 inhibitor (SB02024 or SAR405) treated B16-F10 (Fig. 12A) or CT26 (12B) tumour- bearing mice by ELISA reported in pg/ml. The data are reported as the average of 10 mice per group.

Figure 13 is a graph showing the compound concentration in plasma isolated from blood of B16F10 tumour-bearing mice treated with either SB02024 or SAR405 at the indicated times after dose.

Figures 14A-B illustrate the effect of vehicle or Vps34 inhibitor (SB02024 and SAR405) treatment on tumour progression in B16-F10 (14A) or CT26 (14B) tumour-bearing mice, represented as tumour growth (left graph) or tumour weight at day 17 (right graph).

Figures 15A-B illustrate the effect of vehicle and isotype control, aPD-L1 or aPD1 treatment on tumour progression in B16-F10 (Fig. 15A) or CT26 (15B) tumour-bearing mice, represented as tumour growth (left graph) or tumour weight at day 17 (right graph).

Figure 16A shows the tumour growth curves of B16-F10 tumour-bearing mice treated with SB02024 (left graph) or SAR405 (right graph) together with isotype control, or together with aPD-L1 or aPD1. Figure 16B shows the tumor weight (in grams“g”) on day 17. Figure 17A shows the tumour growth curves of CT26 tumour-bearing mice treated with SB02024 (left graph) or SAR405 (right graph) together with isotype control (triangles), or together with aPD-L1 (grey diamonds/circles) or aPD1 (black diamonds/circles). Figure 17B shows the tumour weight (in grams“g”) on day 17.

Figure 18A is a graph showing the growth curve of control (“sh-CT”) or shVps34 (“sh-Vps34”) B16F10 tumours (n=10, average of 3 independent experiments). Tumour burden was assessed by caliper measurements on days 9, 11 , 13, 15, 17 and 19 after inoculation. Figure 18B shows the knock down by western blot and quantification of the Vps34 band intensity in shVps34 (“Vps34”) versus sh-control (“CT”) B16-F10 cells.

Figure 19 is a graph illustrating flow cytometry quantification of infiltrating CD45⁺ leukocytes (gated in live cells) in control (“sh-CT”) or shVps34 (“sh- Vps34”) B16F10 tumours on day 15.

Figure 20 is a graph showing quantification of the percent of different subpopulations of anti-tumour immune effector cells infiltrating control (“sh- CT”) or shVps34 (“sh-Vps34”). Indicated cells are NK cells (NK), CD8 T cells (CD8), CD4 effector T cells (CD4 eff), dendritic cells (DC), type 1

macrophages (M1 ).

Figure 21 is a graph showing quantification of the percent of PD1 ⁺ NK, CD8 and CD4 effector T cells (CD4 eff) in control (“sh-CT”) or shVps34 (“sh- Vps34”) B16-F10 tumours.

Figure 22 is a graph showing quantification of the percent of PD-L1 ⁺ cells in CD45 tumour cells, CD11 b⁺ cells, F480⁺, dendritic cells (DC) and Myeloid derived suppressor cells (MDSC) in control (“sh-CT”) or shVps34 (“sh- Vps34”) B16-F10 tumours. In terms of quantifying the immune cells infiltrating the tumours, the defined subpopulations were gated in live cells. Each dot represents one tumour in all of the panels. The data are reported as the average of 5 mice per group.

Figure 23 shows the expression level CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11 mRNA, in shVps34 (“sh-Vps34”) B16-F10 cells reported as FC compared with control (“sh-CT”) B16-F10 cells.

Figure 24 shows two graphs illustrating quantification of the secreted CCL5 (left) or CXCL10 protein levels (right) in control (“sh-CT”) or shVps34 (“sh- Vps34”) B16-F10 cell supernatant by ELISA reported in pg/ml. The data are reported as the average of 3-6 independent experiments.

Figure 25 shows two graphs illustrating quantification of the secreted CCL5 (left) or CXCL10 protein levels (right) in control (“shCT”) or shVps34

(“shVPS34”) B16-F10 tumour plasma by ELISA reported in pg/ml

standardized to excised tumour weight in grams (g).

Figure 26 is a graph illustrating quantification of the secreted IFNy in control (“shCT”) or shVps34 (“shVPS34”) B16-F10 tumour plasma by ELISA reported in pg/ml standardized to excised tumour weight in grams (g).

Figure 27 shows two graphs illustrating quantification of the secreted CCL5 (left) or CXCL10 protein levels (right) in control (“shCT”) or shVps34

(“shVPS34”) B16-F10 blood plasma from tumour bearing mice by ELISA reported in pg/ml. The data are reported as the average of 10 mice per group

Figure 28 is a graph showing the growth curve of vehicle or Vps34 inhibitor (SB02024 and SAR405) treated Genetic Engineered Modified Mice (GEMM) melanoma model YUMM1.7 (10 mice/group).

Figure 29 is a graph illustrating the expression level of CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11 mRNA in Vps34 inhibitor (SB02024, middle bar, and SAR405, right bar) treated GEMM cells, reported as FC compared with vehicle treated GEMM cells (left bar).

Figure 30 shows two graphs illustrating quantification of the secreted CCL5 and CXCL10 protein levels present in the supernatant of untreated (left bar) or Vps34 inhibitor (SB02024, middle bar, or SAR405, right bar) treated GEMM cells reported in pg/ml. The data are reported as the average of 3-6 independent experiments.

Fig. 31 A is a graph illustrating the protein levels of Vps34, p62, LC3-I and LC3-II and Actin. Fig. 31 B illustrates the quantification of the mRNA levels of CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11 in siVps34 (“si-Vps34”) B16-F10 cells reported as FC compared with si-CT.

Figure 32 shows two graphs illustrating quantification of the secreted CCL5 (left) and CXCL10 (right) protein levels, present in the supernatant of si- Control (“siCT”) or siVps34 B16-F10 cells reported in pg/ml.

Figure 33 is a graph showing the growth curve of vehicle or Vps34 inhibitor (SB02024 and SAR405) treated RENCA Clear Cell Carcinoma model (10 mice/group).

Figure 34 is a graph illustrating the expression level of CCL5 mRNA in Vps34 inhibitor (SB02024 and SAR405) treated Me30966 cells, reported as FC compared with untreated cells (left graph) and quantification of the secreted CCL5 protein levels, present in the supernatant of untreated or Vps34 inhibitor (SB02024 or SAR405) treated Me30966 cells in pg/ml (right graph). The order from left to right of illustrated bars is: cells treated with DMSO, 0.5 mM of SB02024, 2 mM of SB02024, 0.5 mM of SAR405, and 2 mM of SAR405.

Figure 35 is a graph illustrating the expression level of CXCL10 mRNA in Vps34 inhibitor (SB02024 and SAR405) treated Me30966 cells, reported as FC compared with untreated cells (left graph) and quantification of the secreted CXCL10 protein levels, present in the supernatant of untreated or Vps34 inhibitor (SB02024 or SAR405) treated Me30966 cells in pg/ml (right graph). The order from left to right of illustrated bars is: cells treated with DMSO, 0.5 mM of SB02024, 2 mM of SB02024, 0.5 mM of SAR405, and 2 mM of SAR405.

Figure 36 is a graph illustrating the expression level of CCL5 mRNA in Vps34 inhibitor (SB02024 and SAR405) treated Mel501 cells, reported as FC compared with untreated cells (left graph) and quantification of the secreted CCL5 protein levels, present in the supernatant of untreated or Vps34 inhibitor (SB02024 or SAR405) treated Mel501 cells in pg/ml (right graph). The order from left to right of illustrated bars is: cells treated with DMSO, 0.5 mM of SB02024, 2 mM of SB02024, 0.5 mM of SAR405, and 2 mM of SAR405.

Figure 37 shows survival curves for B16F10 tumour-bearing animals treated either with Vps34 inhibitor (SB02024 or SAR405, upper left graph) or isotype control alone (upper right graph), or with each Vps34 inhibitor in combination with aPD-L1 or aPD1 (SB02024, lower left graph; SAR405, lower right graph) Animals were sacrificed when tumor reached a size of 1000 mm³, accounting for an event in the cascade plot.

In the figures, the error bar represents the SEM. ^* p <0.05; ^** p <0.01 ; ^*** p <0.001 ; ns: not significant; defined using an unpaired, two-tailed Student’s t- test.

DETAILED DESCRIPTION

The great promise of anti-PD-1/PD-L1 -based cancer immunotherapy is observed in a small subset of patients, while the majority of them do not respond, or only partially responds, to this therapy. As mentioned above, durable clinical responses using anti-PD-1/PD-L1 -based immune checkpoint blockade therapy have been associated with T cell-inflamed tumour microenvironment favoring the infiltration of functional cytotoxic T

lymphocytes (CTL). Obviously, T cells would not be able to control tumour growth if they fail to enter the tumour microenvironment. It is therefore believed that driving cytotoxic immune cells into the tumour bed could significantly improve the efficacy of immune checkpoint inhibitors, notably in patients displaying an immune desert tumour phenotype.

The PI3K Class III, vacuolar protein sorting 34 (Vps34, PIK3C3) forms a heterodimer with its regulatory subunit p150 (Vps15) and this dimer takes part in several complexes regulating vesicular trafficking events such

as autophagy, endocytosis, exocytosis and micropinocytosis

As shown in the examples, inhibiting autophagy proteins complex Beclin- 1A/ps34 induces inflammatory tumour microenvironment favoring the infiltration of T cells into the tumour bed. Thus, inhibiting Vps34 makes melanoma tumours eligible for, and thus improves the therapeutic benefit of, anti-PD-1/PD-L1 immunotherapy. Hence, cancer patients that would normally be non-responders to immune checkpoint blockade immunotherapy can still be successfully treated with a combination therapy, if an immunostimulating treatment with a Vps34 inhibitor is found to be effective.

Now, the present inventors have found that certain compounds can be surprisingly useful as biomarkers for determining, evaluating and/or predicting the effect of an autophagy inhibitor, in particular a Vps34 inhibitor, in vitro, ex vivo as well as in vivo. For instance, the biomarkers identified by the present inventors can be used to determine, evaluate and/or predict the immuno- stimulating effect of an autophagy inhibitor, such as a Vps34 inhibitor, based on a blood sample from a subject, for example a cancer patient, that has received treatment with said inhibitor. The information provided by

assessment of the biomarker level may serve as a guide for further treatment. In particular, where assessment of the biomarker(s) indicates an

immunostimulating effect on cancer cells, a cancer patient may

advantageously be subjected to further treatment with immunotherapy, with greater expectation of success compared to a situation where a patient is subjected to immunotherapy without prior or simultaneous treatment with an autophagy inhibitor, or without prior evaluation of the effectiveness of a treatment with an autophagy inhibitor.

As used herein,“autophagy inhibitor” refers to a compound capable of hampering the ability of cells to form or mature autophagosomes.

The biomarkers may be used for determining, evaluating and/or predicting the effect of an autophagy inhibitor, in particular a vps34 inhibitor, in cells in vitro ; or in cells, tumours, tissues, organs ex vivo or in vivo, i.e. in the body of a human or animal.

Cells in vitro may comprise established cell lines or a cell culture originating from cells obtained from a living subject, such as a cancer patient. The cells may be cancer cells. Examples of cell lines representing various types of cancer cells include Renca, BT-20, BT-474, HCC70, MCF-7, MCF10A, MDA- MB-231 , OVCAR-3, SK-OV-3, B16F10, 769-P, 786-0, PC-3, DU-145, PANC- 1 , KP4, KARPAS 299, SKBR3, Calu6, SCC-9, SCC-15, PC9, HCC827, Red , H 1299, H 1975, DMS 114, NCI-H446, NCI-H69, NCI-H82, SHP-77, MG-63, T47D, DLD1 , Kasumi-1 , THP-1 , OCI-M1 , U937, Mel501 , Me30966 and SW- 780.

The biomarker(s) described herein may be used in an in vitro method for assessment of the immunostimulatory effect of a Vps34 inhibitor, where the immunostimulation has been effected in vitro or in vivo. For example, in embodiments, the biomarker level may be determined using an in vitro method as described below based on a clinical test sample obtained from a living subject, in particular a cancer patient. The information gained from determination of the biomarker level in a clinical test sample and comparison thereof to a reference is very useful as it may form a basis for more accurate decisions regarding further treatment, and may hence improve the chances of successful therapy. Furthermore, determination of the level of the

biomarker(s) in a test sample obtained from a subject prior to treatment with Vps34 inhibitor can even be used to distinguish a sub-population of subjects, typically a sub-population of cancer patients, that have abnormally low levels of said biomarker(s) and which are potentially more responsive to treatment with a Vps34 inhibitor, in relation to a general population.

In other embodiments, the biomarker level may be determined in a sample obtained from an in vitro source, such as a cell culture. Determination of the immunostimulatory effect using the biomarker(s) described herein in cell cultures, in particular those based on established cell lines, may be useful during research and development of new therapies based on or including treatment with an autophagy inhibitor, such as a Vps34 inhibitor. The biomarker(s) of the present invention may thus greatly facilitate the

identification of drug candidates that can be effective in vivo.

Furthermore, the biomarker(s) described herein may be useful as an inclusion criterion during a clinical trial, such as a clinical trial evaluating the

effectiveness of a Vps34 inhibitor. For example, prior to admittance of a subject into the trial, determination of the level the biomarker(s) in a test sample obtained from the subject prior to treatment with Vps34 inhibitor can be used for identifying subjects suitable for inclusion in the study and/or to identify subjects that are less likely to be responsive to the treatment subject to the trial and therefore unsuitable to include in a study.

The level of the biomarker(s) in a sample may refer to the concentration of the biomarker compound, typically protein, in the sample. For instance, the level of a biomarker in the sample means the protein concentration of the biomarker in a fluid sample expressed, for instance, as ng/ml. Alternatively, “level” may refer to mRNA expression of a biomarker as determined by any suitable conventional RNA extraction and quantification method.The level of the biomarker(s) in a sample may be determined using any suitable quantitative analytical methods known to persons skilled in the art. For instance, immunoassay techniques using such an assay format are well known, including single-site and two-site or "sandwich" assays of the non- competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a

target biomarker.

Examples of suitable methods include direct and indirect ELISA and variants thereof known to persons of skill in the art, such as electrochemiluminescent labels conjugated to detection antibodies (e.g. MSD assays) and the like.

Examples of suitable methods also include RNA-seq, qPCR and variants thereof known to persons of skill in the art, such as RT-qPCR.

The sample in which the level of biomarker(s) is determined or detected is typically a biological sample maintained in vitro and originating from an in vitro or in vivo source, optionally a living subject. Examples of suitable sample materials include cell culture supernatant, cell lysate, cell secreted vesicles, tumour plasma, tumour lysate, biopsies, and bodily fluids such as blood, blood plasma, and urine. In embodiments, the sample is blood plasma.

As used herein,“a (the) sample” may refer to a plurality of samples which may optionally be obtained over an extended time period, for instance one or more hours, days, or weeks.

As recognized by a person of skill in the art, a sample may require

preparation or processing prior to determination of the biomarker level, such as dilution, concentration, purification and/or separation of sample

components, as recognized by persons of skill in the art.

To provide information that is useful for research, development or clinical purposes as described herein, the level of biomarker(s) determined in a test sample is compared to a reference level of the biomarker. The reference level may be a general threshold level, or the reference level may be determined based on one or more reference samples, optionally originating from the same subject prior to treatment with a Vps34 inhibitor. The term“level” may refer to an absolute amount, or a relative amount, e.g. a concentration, or a value resulting from a detectable signal such as an intensity signal. As used herein,“a level” or“the level” may refer to a single measurement, or to an average value calculated from a plurality of

measurements of a single sample, or to an average value based on a plurality of samples, and optionally based on a plurality of measurements of each sample.

The term "comparing" as used herein refers to comparing the level of the biomarker in the test sample with the reference level of the biomarker. It is to be understood that comparing as used herein usually refers to a

comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from the biomarker in a sample is compared to a reference value based on the same type of intensity signal. The comparison may be carried out manually or computer-assisted. Thus, the comparison may be carried out by a computing device. The value of the determined or detected level of the biomarker in the sample and the reference level can be, e.g., compared to each other and the said comparison can be automatically carried out by a computer program executing an algorithm for the comparison.

In embodiments, the level of more than one biomarker is determined in the test sample, and each compared to a reference level of the same biomarker, each of which may be, independently, either a general threshold level or a reference level determined in a reference sample. Hence, in embodiments a panel of at least two biomarkers as described herein is analysed.

In embodiments using a plurality, or panel, of biomarkers, a combination of predetermined threshold levels and actual reference sample levels may be used. In embodiments, a biomarker level that is increased by at least 50 % relative to the reference level is considered to indicate an immunostimulatory effect.

In particular, an increase of at least 30 %, at least 50 %, at least 75 %, at least 100 %, at least 150 %, at least 200 %, or at least 300 % is considered to indicate an immunostimulatory effect. It may be noted that the degree of increase may vary between subjects, but an increase of at least 30 % is presently believed to be indicative of an immunostimulatory effect in most subjects experiencing such an effect.

The increase in the level of biomarker is preferably a statistically significant increase as determined using a relevant statistical method, e.g. a Student’s t- test with a p-value of less than 0.05 (^*p<0.05), which is also described in the Examples below.

In embodiments, the reference to which the level of the biomarker(s) is compared may be a general, predetermined threshold value based for instance on a normal expected level of the biomarker(s) on a healthy population, or a population not suffering from cancer. Alternatively, the threshold value may be set based on the average level of the biomarker(s) in a population of cancer patients.

Alternatively, in embodiments, a reference sample in which the level of biomarker(s) is determined may be a biological sample maintained in vitro and originating from an in vitro or in vivo source, optionally a living subject. Examples of suitable sample materials include cell culture supernatant, cell lysate, cell secreted vesicles , tumour plasma, tumour lysate, biopsies , and bodily fluids such as blood, blood plasma, and urine.

Typically, when a biological sample is used for determining the reference biomarker level, this reference sample is typically of the same type as the test sample. A reference sample is typically obtained or extracted from the source prior to exposure to a Vps34 inhibitor. Thus, in the case where the sample source is a living subject, the reference sample is typically obtained from the subject prior to treatment with a Vps34 inhibitor, e.g. prior to administration of the Vps34 inhibitor.

The biomarker(s) of the present invention may be a cytokine, such as one or more selected from the group consisting of IL-1 a/IL-1 F1 , IL-1 b/IL-l F2, , IL- 1 ra/IL-1 F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-1 1 , IL-12, L-13, IL-15, IL- 17A, IL-22, IL-23, IL-27, IL-28A/B, IL-33, IFNp, IFNy, TGFa, TGF , IL17RC, IL17RD, CX3CL1/Fractalkine, CXCL1/KC, CXCL2/MIP-2, CXCL9/MIG, CXCL10/IP-10, CXCL1 1/l-TAC, CXCL13/BLC/BCA-1 , CXCL16

CCL2/JE/MCP-1 , CCL3/CCL4/MIP-1 a/ , CCL5/RANTES, CCL6/C10,

CCL1 1/Eotaxin, CCL12/MCP-5, CCL17/TARC, CCL19/MIP-3 , CCL20/MIP- 3a, CCL21/6Ckine, CCL22/MDC, and CXCL5.

In some embodiments, the biomarker(s) may comprise at least one cytokine selected from the group consisting of IL-1 a/IL-1 F1 , IL-1 b/IL-l F2, , IL-1 ra/IL- 1 F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-1 1 , IL-12, L-13, IL-15, IL-17A, IL-22, IL-23, IL-27, IL-28A/B, IL-33, IRNb, IFNy, TGFa, TGF , IL17RC, IL17RD.

In some embodiments, the biomarker(s) may comprise at least one chemokine selected from the group consisting of CX3CL1/Fractalkine, CXCL1/KC, CXCL2/MIP-2, CXCL9/MIG, CXCL10/IP-10, CXCL1 1/l-TAC, and CXCL13/BLC/BCA-1 , CXCL16 CCL2/JE/MCP-1 , CCL3/CCL4/MIP-1 a/b, CCL5/RANTES, CCL6/C10, CCL1 1/Eotaxin, CCL12/MCP-5, CCL17/TARC, OOί19/MIR-3b, CCL20/MIP-3a, CCL21/6Ckine, CCL22/MDC, and CXCL5.

In embodiments, the biomarker(s) may be one or more selected from the group consisting of CCL2, CCL3, CCL4, CCL5 (also known as RANTES), CXCL9, CXCL10, CXCL1 1 and IFNy. For instance, the biomarker may be CCL5 and/or CXCL10. In embodiments, the biomarker(s) may be one or more selected from the group consisting of CCL2, CCL3, CCL4, CCL5 (also known as RANTES), CXCL9, CXCL10 and IFNy. For instance, the biomarker may be CCL5 and/or CXCL10.

In some embodiments of the invention, a panel of two or more of the biomarkers disclosed herein is used. For instance, a panel of biomarkers may include at least CCL5 and CXCL10, and possibly further biomarkers optionally selected from among these disclosed herein.

The Vps34 inhibitor whose effectiveness is evaluated may be any known Vps34 inhibitor.“Vps34 inhibitor” is defined above.

In embodiments, the Vps34 inhibitor may be selected from the compounds listed in Table I below.

Table I. Examples of Vps34 inhibitors

In embodiments, the Vps34 inhibitor is not (2S)-1 -[(5-chloro-3-pyridyl)methyl]- 8-[(3R)-3-methylmorpholin-4-yl]-2-(thfluoromethyl)-3,4-dihydro-2H- pyrimido[1 ,2-a]pyhmidin-6-one (SAR405).

In embodiments, the Vps34 inhibitor may be selected from the group consisting of: N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]acetamide; 4-(2-methyl-1 H-pyrrolo[2,3-b]pyridin-4-yl)-6-morpholino- 1 H-pyridin-2-one; 4-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-6-[3- (trifluoromethyl)morpholin-4-yl]-1 H-pyridin-2-one; 4-(1 H-pyrazolo[3,4- b]pyridin-4-yl)-6-[2-(trifluoromethyl)-1 -piperidyl]-1 H-pyridin-2-one; N-[4-[2-oxo- 6-[3-(trifluoromethyl)morpholin-4-yl]-1 H-pyridin-4-yl]-2-pyridyl]acetamide; 4- [(3R)-3-methylmorpholin-4-yl]-6-[4-(2-methylpyrazol-3-yl)sulfonyl-2- (trifluoromethyl)piperazin-l -yl]-1 H-pyridin-2-one; 6-[4-cyclopropylsulfonyl-2- (trifluoromethyl)piperazin-1 -yl]-4-[(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2- one; 4-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-6-[2- (trifluoromethyl)phenyl]-1 H-pyridin-2-one; methyl N-[4-[2-(2-chlorophenyl)-6- oxo-1 H-pyridin-4-yl]-2-pyridyl]carbamate; 4-[(3R)-3-methylmorpholin-4-yl]-6- [(2R)-2-(trifluoromethyl)-1 -piperidyl]-1 H-pyridin-2-one; 4-[(3R)-3- methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)pyrrolidin-1 -yl]-1 H-pyridin-2- one; 4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-2- one; 6-[2-(3-methoxyphenyl)pyrrolidin-1 -yl]-4-[(3R)-3-methylmorpholin-4-yl]- 1 H-pyridin-2-one; 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-phenyl-1 - piperidyl]-1 H-pyridin-2-one; 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2S)-2- (trifluoromethyl)-l -piperidyl]-1 H-pyridin-2-one; 6-(2-chlorophenyl)-1 -methyl-4- (3-methylmorpholin-4-yl)pyridin-2-one; 4-[(3R)-3-methylmorpholin-4-yl]-6-[2- (trifluoromethyl)phenyl]-1 H-pyridin-2-one; 6-(2-chlorophenyl)-4-[(3R)-3- methylmorpholin-4-yl]-1 H-pyridin-2-one; and 6-(2-chloro-5-fluoro-phenyl)-4- [(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2-one.

In embodiments, the Vps34 inhibitor may be selected from the group consisting of 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-phenyl-1 -piperidyl]- 1 H-pyridin-2-one, 4-[(3R)-3-methylmorpholin-4-yl]-6-[2- (trifluoromethyl)phenyl]-1 H-pyridin-2-one, 4-[(3R)-3-methylmorpholin-4-yl]-6- [(2S)-2-(trifluoromethyl)-1 -piperidyl]-1 H-pyridin-2-one, 4-(1 H-pyrazolo[3,4- b]pyridin-4-yl)-6-[2-(trifluoromethyl)-1 -piperidyl]-1 H-pyridin-2-one, methyl N-[4- [2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2-pyridyl]carbamate, 4-[(3R)-3- methylmorpholin-4-yl]-6-[4-pyrrolidin-1 -ylsulfonyl-2-(trifluoromethyl)piperazin- 1 -yl]-1 H-pyridin-2-one, (2S)-1 -[(5-chloro-3-pyridyl)methyl]-8-[(3R)-3- methylmorpholin-4-yl]-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1 ,2- a]pyrimidin-6-one, and 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2- (trifluoromethyl)-l -piperidyl]-1 H-pyridin-2-one.

In embodiments, the Vps34 inhibitor is 4-[(3R)-3-methylmorpholin-4-yl]-6- [(2R)-2-(trifluoromethyl)-1 -piperidyl]-1 H-pyridin-2-one.

The immunotherapy may be an immune checkpoint blockade-based therapy. A non-limiting example is anti-PD-1 or PD-L1 based immunotherapy, and may optionally comprise treatment with an agent selected from the group consisting of Nivolumab, Pembrolizumab, Atezolizumab, Avelumab,

Durvalumab, Cemiplimab, Tislelizumab, and Sintilimab.Another example is macrophage immune checkpoint based immunotherapy, e.g. involving treatment with anti-CD47 antibody or anti-SIRPalpha antibody. Alternatively or additionally the immunotherapy may comprise one or more of the following: an anti-CTLA-4 based immunotherapy (e.g. including treatment with an agent selected from Ipilimumab and Tremelimumab), treatment with an oncolytic virus (e.g. selected from Oncorine, Talimogene laherparepvec and

Pelareore), a CAR-T cell therapy (e.g. including treatment with an agent selected from Tisagenlecleucel or Axicabtagene ciloleucel), treatment with a CD3-targeted bispecific antibody (e.g. Blinatumomab or duvortuxizumab), an anti-NKG2A based immunotherapy (e.g. comprising treatment with

Monalizumab), an anti-KIR based immunotherapy (e.g. comprising treatment with IPH4102 or Lirilumab), a STING based immunotherapy (comprising treatment with a STING agonist, such as a cyclic nucleotide, e.g. selected from MK-1454, ADU-S100 and SB 1 1285). The present invention is useful in the field of cancer research and treatment. The cancer may be a tumorigenic cancer or a non-tumorous cancer. Cancer forms contemplated for application of the present invention include, but are not limited to, breast cancer, including triple negative breast cancer, pancreas cancer, leukemia, melanoma, bladder cancer, kidney cancer, head and neck squamous carcinoma, lung cancer and colorectal cancer (CRC). However, the invention may be applied with any form of cancer that would desirable to treat by immunotherapy. Hence, the invention may be useful for evaluating whether a particular form of cancer or a particular cancer-afflicted subject may potentially be responsive to treatment with immunotherapy.

The examples below show that pharmacologically inhibiting the protein Vps34 in B16-F10 and GEMM melanoma, colorectal cancer model (CT26) and Clear Cell carcinoma kidney model (RENCA) inhibits tumour growth by inducing the infiltration of CD8⁺ T cells. Similar results were achieved in B16-F10 cancer model when Vps34 expression was diminished significantly by the use of shRNA designed to block Vps34 expression, which reduced the protein levels to approxiamtely 40% (genetic inhibition of Vps34 function). In fact, pharmacological or genetic inhibition of Vps34 was found to have a broad impact on the immune landscape of melanoma tumours by inducing infiltration of NK cells and infiltration of CD8⁺ T cells into the tumour bed. It was revealed that such infiltration is mechanistically related to the impact of inhibiting autophagy on the reprograming of a cold immune desert tumour microenvironment to an inflamed immune receptive one.

In autophagy defective melanoma and colorectal tumours, a T cell inflamed tumour microenvironment was established, characterized by the upregulation of CCL5 and CXCL10 chemokines favoring the infiltration of CD8⁺ T into the tumour bed. Significantly higher levels of CCL5 compared to untreated animals where found in the blood of melanoma- or CRC-bearing mice treated with Vps34 inhibitor (Vps34i), and CXCL10 was also significantly higher in blood from melanoma- bearing mice. Furthermore, high levels of CCL5, CXCL10 and IFNy were found in tumour plasma from melanoma- or CRC- bearing mice treated with Vps34 inhibitor, indicating that these cytokines may constitute valuable biomarkers predicting the in vivo response to Vps34i.

It is furthermore shown that autophagy defective tumour cells displayed increased expression of PD-L1 which is mechanistically related to the high level of IFNy released in the tumour microenvironment and the activation of JAK2/STAT1 signaling pathway in tumour cells. It is shown that the

therapeutic benefit of anti-PD-1/PD-L1 was significantly improved by combination with Vps34i in melanoma and CRC.

Overall, the examples reveal for the first time that inhibiting Beclin-1 A/ps34 complex makes melanoma and CRC tumours eligible for immune checkpoint blockade-based therapy by creating T cell inflamed tumour microenvironment.

The study represented by present examples below thus provides cutting edge advances on how autophagy inhibition makes tumours eligible for

immunotherapies and establishes the proof-of-concept to set-up innovative clinical trials using selective autophagy inhibitor drugs in combination with immune checkpoint blockades.

EXAMPLES

Example 1. Cell cultures and shVPs34 or siVPs34 transfections

The B16-F10, CT26 and YUMM1.7 GEMM cell lines were purchased from ATCC. The Renca cell line (440321 ) was purchased from CLS. RPMI 1640, DMEM, FBS, and antibiotics were obtained from Life Technologies. Cell lines were cultured in RPMI 1640 or DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1 % Penicillin/Streptomycin at 37°C and 5% C02. To generate shVps34 or siVps34 B16-F10 cells, B16-F10 cells were transfected according to the manufacturer’s protocol with either Control shRNA Lentiviral Particles (sc-108080) or Pik3c3 (Vps34) Lentiviral Particles (sc-62803-V) purchased from Santa Cruz Biotechnology, respectively. Example 2. In vivo tumour growth and mice treatments

C57BL/6 and BALB/C mice (7 weeks old) were purchased from Janvier and housed in pathogen-free conditions for 1 week prior to the experiments.

Mice were injected subcutaneously in the right flank with different cell lines diluted in 100 pi of PBS. The anti-mouse CD8a (clone: 2.43; catalog#:

BE0061 ), anti-mouse PD-1 (clone: 29F.1A12 ; catalog#: BE0273), anti-mouse PD-L1 (clone: 10F.9G2; catalog#: BE0101 ), anti-mouse IFNy (clone: XMG1.2; catalog#: BE0055) and their Rat lgG2b isotype control (clone: LTF-2;

catalog#: BE0090) and rat lgG2a isotype control, anti-trinitrophenol (clone: 2A3; catalog#: BE0089) were purchased from BioXCell and administered as described below.

Vps34i SB02024 was provided by Sprint Bioscience (Sweden) and

administrated to the mice at 20 mg/kg by oral gavage. SAR405 (HY-12481 ; Bio-Connect) was administrated to the mice at 10 mg/kg by oral gavage. Vehicle treatment was performed using methylcellulose(0.5% in water) supplemented with 1 % polysorbate-80.

In B16F10 and CT26 tumour bearing animals, combination with anti- PD-1 or PD-L1 was done on animals treated with vehicle or 20 mg/kg SB02024 or 10 mg/kg SAR405. Animals were injected (i.p.) with 200 pg of either antibody isotype control or anti- PD-1 or anti-PD-L1 every other day (completing a total of 5 doses).

For depletion of CD8⁺ T-cells, B16-F10 animals were pretreated with aCD8 or isotype control 3 days before injection of B16-F10 or CT26 cells as described above. Injection of isotype or CD8 antibody was repeated at days 3, 7, 10, 13 and 16.

Example of typical dosing regime: B16-F10 cells were injected

subcutaneously into the right flank of syngeneic host C57BL/6 mice and 7 days after, when tumour was palpable, animals were dosed by oral gavage with vehicle or Vps34 inhibitor SB02024 or SAR405 at 20 mg/kg and 10 mg/kg respectively, once daily. The mice were sacrificed on days 11 , 14, 17, 19 and 21 for flow cytometry analysis, cryopreservation and protein and RNA extraction (see Examples below). For blood plasma preparation, blood was collected by cardiac puncture of sacrificed animals two hours after vps34i dose, and centrifuged for 10 minutes at 1500g. The supernatant was collected for ELISA.

The mouse experiments were performed according to the instructions and guidelines of Luxembourg Institute of Health and approved by the LIH ethical committee.

The tumours were measured using a calipers along three axes: length (L), width (W) and height (H). Volume was calculated using the following formula: Volume (mm³) = L^*H^*W ^*(p/6) on the indicated days. Mice that did not develop tumours or developed tumours larger than the threshold defined in the in vivo protocols approved by the animal welfare committee of

Luxembourg Institute of Health (volume > 2000 mm³) were excluded. The mice were sacrificed on days 11 , 14, 17, 19 and 21 for flow cytometry analysis, cryopreservation and protein and RNA extraction.

Example 3. Tumour isolation and digestion for cell population analyses.

Tumours were dissected and mechanically dissociated into small, <4-mm fragments with a scalpel, followed by digestion with mouse tumour

dissociation kit (Miltenyi Biotec) for 45 min at 37°C. After single-cell suspensions were obtained, red blood cells were removed by ACK (10-548E, Lonza). The cells were next counted using a Countess Automated Cell Counter (Invitrogen) and blocked for 30 minutes on ice with Fc block

(TruStain fcX™ (anti-mouse CD16/32) Antibody 101320 Biolegend). The samples were first stained for surface markers for lymphoid and myeloid immune populations followed by intracellular staining. For FoxP3 and intracellular staining, True-Nuclear™ Transcription Factor Buffer Set 424401 Biolegend was used according to the manufacturer’s recommended protocol.

Example 4. Flow cytometry

The following antibodies were purchased from Biolegend: FITC anti-mouse CD45, Brilliant Violet 785 anti-mouse CD3, APC anti-mouse CD8a, APC/Fire 750 anti-mouse CD4, PE/Cy7 anti-mouse CD49b (pan-NK cells), PE/Cy7 anti-mouse NK-1.1 Antibody, Brilliant Violet 605 anti-mouse CD69, PE/Cy5 anti-mouse CD25, Brilliant Violet 421 anti-mouse FOXP3, PE/Dazzle 594 anti-mouse CD279 (PD-1 ), Brilliant Violet 785 anti-mouse/human CD11 b,

APC anti-mouse F4/80, PE/Cy5 anti-mouse CD11 c, PE/Cy7 anti-mouse Ly- 6G, APC/Fire 750 anti-mouse Ly-6C, Brilliant Violet 605 anti-mouse CD206 (MMR), and Brilliant Violet 421 anti-mouse CD274 (B7-H 1 , PD-L1 ).

LIVE/DEAD Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific) was used as a viability dye. Single stains were performed for compensation controls, FMO controls to check for fluorescence spread and isotype controls were used to determine the level of non-specific binding.

Example 5. RNA extraction and reverse transcriptase-polymerase chain reaction

Total RNAs were extracted using the miRCURY RNA isolation kit (300110; Exiqon) and quantified using Nanodrop. RNA (200 ng) from each sample was reverse transcribed using RT reaction mix (Eurogentec). The reverse transcription was performed at 48°C for 30 min. The resulting cDNA was subjected to amplification by quantitative PCR using power SYBR green PCR master mix (Life Technologies). The RPL13 gene encoding Ribosomal Protein L13 was used as an internal control. For mouse RPL13, the following primers were used: forward 5’ GGA GGG GCA GGT TCT GGT AT-3’ and reverse 5’-TGT TGA TGC CTT CAC AGC GT-3’. For mouse IFNy, the following primers were used: forward 5’-GAT-GCA-TTC-ATG-AGT-ATT-GCC- 3’ and reverse 5’-GTG-GAC-CAC-TCG-GAT-GAG-CTC-GG-3’. For mouse cxcHO, the following primers were used: forward 5’-CCA CGT GTT GAG ATC ATT GCC-3’ and reverse 5’-GAG GCT CTC TGC TGT CCA TC-3’. For mouse ccl5, the following primers were used: forward 5’-CTG CTG CTT TGC CTA CCT CT-3’ and reverse 5’-CGA GTG ACA AAC ACG ACT GC-3’.

Example 6. ELISA from in vitro and in vivo samples

For tumour plasma preparation, tumours were dissociated in DMEM medium, then centrifuged to collect the supernatant; the supernatant was concentrated with Protein Concentrator PES, 3K MWCO (88526, Fisher Scientific), according to the manufacturer’s protocol. For blood plasma preparation, blood was collected by cardiac puncture before mice sacrifice 2 hours after last dose, and centrifuged for 10 minutes at 1500g, and the supernatant collected for ELISA. For measurements of protein levels of CCL5 and CXCL10 from in vitro cell supernatants, cells were plated in 6-well dishes, and treated with SB02024 (5mM), SAR405 (10mM) for 48 hours. The last 24 hours, cells were deprived from serum.

CCL5, CXCL10 and IFNy were quantified using mouse CCL5/RANTES DuoSet ELISA (DY478-05; R&D Systems), mouse CXCL10/IP-10/CRG-2 DuoSet ELISA (DY466-05; R&D Systems), cxcl10/IP-10/CRG-2 ELISA kit (NBP1 -92665, Novus Biologicals) and Mouse IFN-gamma DuoSet ELISA (DY485-05; R&D Systems).

Example 7: Vps34 inhibitor (Vps34i) treatment of B16-F10 or CT26 tumour bearing mice improves their response to anti-PD1/PD-L1 -based

immunotherapy

It was evaluated whether Vps34i treatment could improve the response of B16-F10 and CT26 tumours to anti-PD1 or anti-PD-L1 therapy by assessing the impact of this treatment on the tumour volume. B16-F10 or CT26 tumour bearing mice were treated with vehicle or Vps34 inhibitor, administered by oral gavage, either 20 mg/kg or 10 mg/kg of SB02024 or SAR405,

respectively, at day 8 and dosed once daily until day 17. At day 9, 11 , 13, 15 and 17, animals were injected intraperitoneally either with anti-PD1 (aPD1 ), anti-PD-L1 (aPD-L1 ) or isotype control. Example 8: Vps34 inhibitor (Vps34i) treatment of cancer cell lines and measurement of CCL5 and CXCL10

A number of cell lines, as shown in Table II, were incubated with different Vps34i to determine modulation of CCL5 and CXCL10 secretion.

Supernatants of cells treated for 24 hours with Vps34i or DMSO (control), as shown in Table III and Table IV, were analysed with Meso Scale Discovery technology. In short, a biotin labelled antibody against the chemokine of interest was added into a MSD Gold Small streptoavidin coated plate (MSD), and supernatant was added. After equilibration time, a Sulfo-Tag conjugated detection antibody was added, allowed to bind and signal in form of electrochemoluminescence was measured. Results were normalised versus the DMSO treated cells.

Table II. Human cancer cell lines used in Example 8

Table III. Effect on secretion of CCL5 and CXCL10 protein upon treatment of Vps34i in human cancer cell lines

^# Effect on secretion of CCL5 anc CXCL10 protein is s nown as ratio of Vps34i treated cells versus untreated (DMSO contro cells.

* Cellular potency, represented as plC50, measured according to the method described in for example W02017140841 and

WO2017140843.

Table IV. Effect on secretion of CCL5 and CXCL10 upon treatment of Vps34i in human melanoma cell line Me30966

^# Effect on secretion of CCL5 and CXCL10 protein is shown as ratio of Vps34i treated cells versus untreated (DMSO control) cells.

* Cellular potency, depicted as plC50, measured according to the method described in for example W02017140841 and WO2017140843.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism software (version 7.00). The error bars represent the standard error of the mean (SEM). The data are represented as the average ± SEM. p values were calculated using an unpaired, two-tailed Student’s ί-test to compare the two groups. A p-value less than 0.05 was considered to be statistically significant. ^* p <0.05; ^** p <0.01 ; ^*** p <0.001 ; ns: not significant.

In vivo study approval

The mice were treated in accordance with European Union guidelines, and the in vivo protocols were approved by the Animal Welfare Society

Luxembourg (agreements n. LECR-2016-02; LECR-2017-02 and LECR- 2017-03). Results

Targeting Vps34 inhibits tumour growth in multiple cancer models

The Examples described above were used to investigate the impact of targeting Vps34 (both genetically and pharmacologically) on tumour growth inhibition (TGI). As described above, genetic targeting Vps34 was achieved by stable transfection of B16-F10 cells with control vector (shCT) or vector encoding Vps34 short hairpin RNA (shVps34). The knock down of Vps34 protein resulted in reduction of the protein levels to approxiamtely 40%

(Figure 18). After inoculation into the left flank of immunocompetent mice, the growth of shCT and shVps34 B16-F10 tumours was monitored. Genetic targeting Vps34 significantly decreased tumour progression (Figure 18A). To assess whether, similar to genetic targeting Vps34, pharmacological inhibition of Vps34 kinase activity also affects the growth and weight of several tumour types, two diverse and selective Vps34 kinase inhibitors (Vps34i) were used, SB02024 and SAR405. Systemic treatment of tumour bearing mice with Vps34i resulted in a significant inhibition of tumour growth in B16-F10 (Figure 1A),GEMM (Figure 28), CT26 (Figure 1 B) and Renca (Figure 33). These results demonstrate that the inhibition of tumour growth resulting from

SB02024 or SAR405 treatment is not restricted to a specific tumour histology or mouse genetic background and it could be broadly applied to multiple tumour models, including melanoma and CRC.

Vps34 targeting enhanced the infiltration of various anti-tumour immune effector cells.

The Examples described above were used to investigate whether the Vps34- dependent antitumour activity is associated with a modulation of the tumour immune landscape. The Examples show that the percentage of live CD45+ cells was significantly increased in shVps34 B16-F10 tumours as compared to shCT B16-F10 tumours (Figure 19). Similarly, Vps34i treatment

significantly increased the percentage of live CD45+ cells in both B16-F10 and CT26 tumours (Figure 19). Flow cytometry comprehensive

immunophenotyping of different cell subpopulations were performed in order to identify and quantify both immune supportive and immune suppressive cell subsets infiltrating B16-F10 tumours genetically defective in Vps34 or pharmacologically treated with Vps34i. A significant increase was observed in the infiltration of immune supportive NK, CD8 T cells, CD4 T effector (eff) cells, dendritic cells (DC) and M1 macrophages in shVps34 and in Vps34i treated B16-F10 tumours as compared to shCT and vehicle-treated controls (Figures 21 and 3, respectively). Similar to B16-F10 tumours, an increased infiltration of NK, CD8+ T cells, DC and M1 macrophages, but not CD4+ T effector cells, was also observed in Vps34i treated CT26 tumours as compared to vehicle-treated control tumours (Figure 3A, 3B).

To assess to what extend the tumour growth inhibition (TGI) is related to the Vps34-dependent increase in infiltration of CD8+ T cells, CD8+ T cells were depleted in vehicle-treated and in Vps34i-treated B16-F10 tumour bearing mice. While the depletion of CD8+ T cells had a slight impact on the growth of weakly infiltrated vehicle-treated tumours, such depletion significantly abolished the effect Vps34i had on growth of B16-F10 melanoma tumours measured as volume (Figure 6). This result highlights that CD8+ T cytotoxic cells are among the the major immune cells controlling the growth of Vps34i- treated B16-F10 tumours.

Inhibition of Vps34 establishes a pro-inflammatory tumour microenvironment When evaluating the expression of a subset of six chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10 and CXCL11 ), previously described to be involved in the recruitment of CD8+ T cells into human melanomas [Harl in et al. (2016) Cancer Res., 69, 3077-3085], it was shown that CCL5 and CXCL10 mRNA and their corresponding secretory protein levels were the only significantly up- regulated in shVps34 B16-F10 melanoma (Figures 23 and 24) and in Vps34i- treated B16-F10 and CT26 (Figures 7A-B and 8A-B) tumour cells as compared to controls. Similarly, siVps34 B16-F10 melanoma cells showed higher levels of CCL5 and CXCL10 mRNA compared to si-control (Figure 31 B) and the release into the supernatant of CCL5 and CXCL10 proteins was also significantly increased (Figure 32). Similar increase in the CCL5 and CXCL10 mRNA and their corresponding secretory protein levels is seen in GEMM cells (Figures 29 and 30). These findings are confirmed in human melanoma cell lines Me30966 and Mel501 treated with Vps34i (Figures 34-35 and 36, respectively). In Me30966 cells incubated with two different concentrations of Vps34i SB02024 or SAR405, the levels of CCL5 and CXCL10 mRNA and CCL5 and CXCL10 proteins secreted to the supernatant were increased in a dose dependent manner compared to vehicle treated cells (Figures 34 and 35). In Mel501 cells incubated with two different concentrations of Vps34i SB02024 or SAR405, the levels of CCL5 mRNA and CCL5 protein secreted to the supernatant were increased in a dose dependent manner compared to vehicle treated cells (Figure 36).

Interestingly, the data shows that higher levels of CCL5, CXCL10 and IFNy are detected in the tumour microenvironment of shVps34 B16-F10 melanoma (Figures 25 and 26) and in Vps34i-treated B16-F10 and CT26 tumours (Figures 10A, 10B and 11 A, 11 B) as compared to controls.

Both CCL5 and CXCL10 mRNA levels were also found to be increased in tumour lysate from Vps34i treated B16-F10 tumours (Figure 9). Treatment with Vps34 inhibitors significantly increased the CCL5 and CXCL10 protein levels in tumour plasma of B16-F10 and CT26 tumour bearing mice (Figures 10A, 10B). The increased expression of CCL5 and CXCL10 could also be observed in the blood plasma of Vps34 inhibitor SB02024 treated B16-F10 and CT26 tumour bearing mice, but not in SAR405 treated mice (Figures 12A, 12B) or shVps34 B16-F10 tumour bearing mice (Figure 27). For the pharmacological inhibition, this can be explained by the different doses (and hence compound exposure, Figure 13) used in the study, where SB02024 was dosed at 20 mg/kg whereas SAR405 was dosed at 10 mg/kg. The difference in pharmacokinetic profiles of SB02024 and SAR045 may also contribute to this difference. For the genetic depletion of Vps34, the reason for not being able to detect an increase of cytokines in blood plasma may be the incomplete deletion of Vps34 achieved in B16-F10 by using the shVps34 constructs (Figure 18B). Still, as shown above, SAR405 induced an increase of CCL5 and CXCL10 at the levels of both mRNA and protein in different systems both in vitro (cells and cells supernatant) and in vivo (tumour and tumour plasma) in a very similar pattern as to SB02024. This together with the observed increase in infiltration as a result of Vps34 inhibition with

SAR405 (similar to the results obtained with SB02024) (Figures 2 and 3), and the effect on tumour growth (Figures 1 A, 1 B), supports the idea of using the increased levels of CCL5 and CXCL10 in blood plasma as a valuable biomarker for the response to Vps34i treatment.

To investigate if this effect of Vps34 inhibition can be extrapolated to other cancer types, a number of human cancer cell lines (Table II) were challenged with a variety of Vps34 inhibitors at 2mM. Increased levels of chemokines were detected in the cellular supernatants (Table III). CCL5 and CXCL10 levels in supernatant were measured (pg/ml), and the ratio versus DMSO control treated cells is shown. In addition, the induction of chemokine secretion in melanoma cell line Me30966 was measured after incubation with several Vps34 inhibitors (Table IV), and the ratio versus DMSO control treated cells is shown. The levels of CCL5 and CXCL10 proteins secreted to the supernatant were increased in a dose dependent manner in cells treated with Vps34i SB02122 compared to vehicle treated cells. The data also points to a correlation between the increase of secreted chemokines and the cellular potency of the Vps34i used, with the compounds displaying higher cellular potency causing a higher increase in secretion compared to control.

In addition, the increase in CCL5 and CXCL10 is most likely generated from the tumour, since in Vps34i-treated non-tumour bearing mice (mice treated once daily with either 20 mg/kg SB02024 or 10 mg/kg SAR405 by oral gavage and sacrificed on day 10) no difference in CCL5 and CXCL10 in the blood plasma were observed.

Altogether, the results indicate that targeting Vps34 by pharmacological inhibition, as exemplified by using SB02024 or SAR405, remarkably induced the secretion of pro-inflammatory chemokine (CCL5, CXCL10 and IFNy) mostly by tumour cells which could subsequently enhance the recruitment of NK, CD8+ T and CD4+ T effector cells into the tumour bed. Vps34i improves the therapeutic benefit of anti-PD-L1/anti-PD-1

Treatment of B16-F10 or CT26 tumour bearing mice with the Vps34 inhibitors SB02024 or SAR405 increased the infiltration of CD45⁺ cells into the tumour bed (Figure 2). Deep immunophenotyping by flow cytometry of immune supportive and immune suppressive immune cells showed a significant increase in the infiltration of NK, T cells (both CD8 and CD4), DC, and M1 (total, inflammatory and resident) (Figure 3A-B). Higher expression level of the early exhaustion maker PD-1 were observed on NK, CD8 and CD4 effector cells infiltrating tumours treated with the Vps34 inhibitor SB02024 or SAR405 compared with vehicle treated control tumours (Figures 4A, 4B). Furthermore, treatment with Vps34 inhibitor SB02024 or SAR405 increased the expression of PD-L1 on the surface of CD45 , CD11 b⁺ myeloid cells and F4/80 macrophages (Figures 5A, 5B). Depletion of CD8⁺ T cells had a little impact on the growth of weakly infiltrated vehicle treated tumours, but it significantly increased the volume of both Vps34 inhibitor treated tumours (SB02024 or SAR405) (Figure 6), highlighting that Vps34 inhibitor treatment inhibited B16-F10 tumour growth by inducing the infiltration of CD8⁺ T cells into the tumour bed. Similarly, genetic ablation of Vps34 in B16-F10 cells resulted in an increased infiltration profile similar to the infiltration changes achieved by pharmacologic inhibition (described above) (Figures 19 and 20). Depletion of Vps34 by shVps34 in B16-F10 cells resulted in an increase of PD-1 expression on NK, CD8 and CD4 effector cells infiltrating tumours compared with sh-control B16-F10 tumours (Figure 21 ). Furthermore Vps34 deletion by shVps34 in B16-F10 increased the expression of PD-L1 on the surface of CD45 , CD11 b⁺ myeloid cells and F4/80 macrophages (Figure 22).

Furthermore, the data shows that anti-PD-L1 or PD-1 monotherapy had no, or marginal, effect on the volume and weight of B16-F10 and CT26 tumours (Figures 15A, 15B). Flowever, combining Vps34i with either anti-PD-L1 or anti-PD1 (Figures 16A, 16B and 17A, 17B) significantly improved their therapeutic benefit as compared to Vps34i monotherapy (Figure 14). The tumour growth inhibition effect of Vps34i/anti-PD-1 combination was observed as early as day 13 of the tumour development, while that of Vps34i/anti-PD- L1 combination was observed at day 17 of the tumour development. This suggests that the therapeutic benefit of Vps34i/anti-PD-1 combination is even greater than that of Vps34i/anti-PD-L1 combination. Further confirmation is provided by the survival curves shown in Figure 37 (as measured by the time until tumours reach a harmful size, in this case 1000mm³ in volume), where anti-PD1 or anti-PL-D1 effect was highly potentiated by combinination with Vps34i.

The person skilled in the art realizes that the present invention by no means is limited to the embodiments described above. On the contrary, many modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practising the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage.

ITEMIZED LIST OF EMBODIMENTS

1. An in vitro method of determining the in vivo immunostimulating effect resulting from pharmacological inhibition of Vps34 by a Vps34 inhibitor, comprising: in vitro analysing a test sample originating from a source to determine a level of at least one biomarker, wherein the at least one biomarker is selected from the group consisting of CCL2, CCL3, CCL4,

CCL5, CXCL9, CXCL10 and IFNy, and preferably selected from CCL5 and CXCL10, wherein an elevated level of said at least one biomarker in said test sample, relative to a reference level, indicates immunostimulation.

2. An in vitro method of determining the in vivo immunostimulating effect resulting from pharmacological inhibition of Vps34 by a Vps34 inhibitor, comprising: in vitro analysing a test sample originating from a source to determine a level of at least one biomarker, wherein the at least one biomarker is selected from the group consisting of CCL2, CCL3, CCL4,

CCL5, CXCL9, CXCL10, CXCL11 and IFNy, and preferably selected from CCL5 and CXCL10, wherein an elevated level of said at least one biomarker in said test sample, relative to a reference level, indicates immunostimulation.

3. The method of item 1 or 2, wherein the mRNA expression level of said at least one biomarker is determined.

4. The method of item 1 or 2, wherein the protein concentration of said at least one biomarker in said sample is determined.

5. The method of any one of the preceding items, comprising i) providing at least one test sample; and

ii) in vitro analysing said test sample to determine the level of said at least one biomarker, wherein an elevated level of said at least one biomarker, relative to a reference level, indcates immunostimulation.

6. The method of any one of the preceding items, wherein the reference level is based on a reference sample from a source that has not been exposed to or subjected to treatment with a Vps34 inhibitor,

7. The method of item 6, comprising in vitro analysing said reference sample and said test sample to determine the level of at least one biomarker in each of said samples, and wherein an elevated level of said at least one biomarker in the test sample relative to the level of the biomarker in the reference sample indicates immunomostimulation in said source.

8. The method of item 5, comprising

i-b) providing a biological test sample originating from a living source subjected to treatment with a Vps34 inhibitor;

ii) in vitro analysing said biological reference sample and said biological test sample to determine the level of at least one biomarker in each of said samples, and wherein an elevated level of said at least one biomarker in the biological test sample relative to the biological reference sample indicates immunostimulation in said source treated with a Vps34 inhibitor. 9. The method of any one of the preceding items, wherein the test sample comprises a material selected from the group consisting of: cell culture supernatant, tumour plasma, and bodily fluids such as blood, blood plasma and urine, and preferably is blood plasma.

10. The method of any one of the preceding items, wherein the reference level is based on a reference sample comprising a material selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine, and preferably is blood plasma.

11. The method of item 9 or 10, wherein the test sample is blood plasma or tumour plasma, preferably blood plasma; and the reference sample is blood plasma or tumour plasma, preferably blood plasma.

12. The method according to any one of the preceding items, wherein said source is a cancer cell or tumour maintained in vitro.

13. A method of determining and/or predicting the immunostimulating effect of treatment with Vps34 inhibitor in a subject afflicted with cancer, wherein said treatment comprises administration of a Vps34 inhibitor to said subject, said method comprising

14. A method of determining and/or predicting the immunostimulating effect of treatment with Vps34 inhibitor in a subject afflicted with cancer, wherein said treatment comprises administration of a Vps34 inhibitor to said subject, said method comprising (i) determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample obtained from said subject, and

15. The method of item 13 or 14, wherein said reference level is based on a reference sample obtained from said subject prior to said treatment.

16. The method of any one of items 13 to 15, wherein the test sample and/or the reference sample is selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine.

17. The method of item 15, wherein the test sample is blood plasma or tumour plasma, preferably blood plasma; and the biological reference sample is blood plasma or tumour plasma, preferably blood plasma.

18. The method of item 15, comprising the steps of

b) optionally administering a Vps34 inhibitor to said subject;

c) providing a test sample originating from said subject after having received said treatment;

19. The method of item 13 or 14, wherein in step i) the mRNA expression level of said at least one biomarker is determined in said test sample. 20. The method of item 13 or 14, wherein in step i) the protein concentration of said at least one biomarker is determined in said test sample.

21. The method of item 15, wherein the mRNA expression level of said at least one biomarker is determined in said reference sample.

22. The method of item 15, wherein the protein concentration of said at least one biomarker is determined in said reference sample.

23. The method of item 13 or 14, wherein the at least one biomarker is selected from CCL5 and CXCL10.

24. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample originating from said subject; and

iii)if the level of said biomarker in said test sample is elevated relative to the reference level, selecting immunotherapy as a further treatment optionally in combination with treatment with the Vps34 inhibitor.

25. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample originating from said; and

iii)if the level of said biomarker in said test sample is elevated relative to the reference level, selecting continued treatment with the Vps34 inhibitor.

26. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor; and

27. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor; and

28. The method of any one of items 24 to 27, wherein step i) further comprises determining the level of said at least one biomarker in a reference sample obtained from said subject prior to treatment with the Vps34 inhibitor, and the predetermined reference level corresponds to the level of the biomarker in the reference sample.

29. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(d) comparing the level of said at least one biomarker determined in step c) to a predetermined reference level; and (e) if the level of said at least one biomarker in the test sample is increased relative to said reference level, further subjecting said subject to immunotherapy treatment.

30. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(d) comparing the level of said at least one biomarker determined in step c) to a predetermined reference level; and

31. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(e) if the level of said at least one biomarker in the test sample is increased relative to said reference level, further subjecting said subject to immunotherapy treatment.

32. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

(c) determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in said test sample; (d) comparing the level of said at least one biomarker determined in step c) to a predetermined reference level; and

33. The method of any one of items 29 to 32, comprising, prior to the step of (a) administering a Vps34 inhibitor to said subject, obtaining a reference sample from said subject, and further comprising determining the level of said at least one biomarker in said reference sample, and wherein said predetermined reference level represents the level of said at least one biomarker determined in said reference sample.

34. The method of item 29 or 31 , wherein said subject is subjected to further treatment with the Vps34 inhibitor simultaneously, intermittently or sequentially with said immunotherapy.

35. The method of any one of items 24, 25, 26, 29, 30, 31 and 32, wherein the mRNA expression level and of said at least one biomarker is determined in said test sample.

36. The method of any one of items 24, 25, 26, 29, 30, 31 and 32, wherein the protein concentration of said at least one biomarker is determined in said test sample.

37. The method of any one of items 28 or 33 wherein the mRNA expression level of said at least one biomarker is determined in said reference sample.

38. The method of item 28 or 33 wherein the protein concentration of said at least one biomarker is determined in said reference sample.

39. The method of any one of items 24, 25, 26, 27, 29, 30, 31 and 32, wherein the biological test sample is selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine.

40. The method of items 28 or 33 wherein the biological reference sample is selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine. 41. The method of item 40, wherein the biological test sample is blood plasma or tumour plasma, preferably blood plasma; and the biological reference sample is blood plasma or tumour plasma, preferably blood plasma.

42. The method of any one of items 24, 26, 29 and 31 ,, wherein said immunotherapy treatment is an immune checkpoint blockade-based therapy.

43. The method of item 42, wherein said immunotherapy is anti-PD-1 or PD-L1 based immunotherapy or macrophage immune checkpoint based immunotherapy.

44. The method of item 43, wherein said anti-PD-1 or PD-L1 based immunotherapy comprises treatment with an agent selected from the group consisting of Nivolumab, Pembrolizumab, Atezolizumab, Avelumab,

Durvalumab, Cemiplimab, Tislelizumab, Sintilimab or said macrophage immune checkpoint based immunotherapy comprises treatment with an agent selected from the group consisting of an anti-CD47 antibody and an anti- SIRPalpha antibody.

45. The method of any one of items 24, 26, 29 and 31 ,, wherein said immunotherapy treatment is an anti-CTLA-4 based immunotherapy.

46. The method of item 45, wherein said anti-CTLA-4 based

immunotherapy comprises treatment with an agent selected from Ipilimumab and Tremelimumab.

47. The method of any one of items 24, 26, 29 and 31 , wherein said immunotherapy treatment comprises treatment with an oncolytic virus.

48. The method of item 47, wherein said oncolytic virus is selected from Oncorine, Talimogene laherparepvec and Pelareorep.

49. The method of any one of items 24, 26, 29 and 31 ,, wherein said immunotherapy treatment is a CAR-T cell therapy.

50. The method of item 49, wherein said CAR-T cell therapy comprises treatment with Tisagenlecleucel or Axicabtagene ciloleucel.

51. The method of any one of items 24, 26, 29 and 31 , wherein said immunotherapy treatment comprises treatment with a CD3-targeted bispecific antibody. 52. The method of item 51 , wherein said CD3-targeted bispecific antibody is Blinatumomab or duvortuxizumab.

53. The method of any one of items 24, 26, 29 and 31 , wherein said immunotherapy treatment is an anti-NKG2A based immunotherapy.

54. The method of item 53, wherein said anti-NKG2A based

immunotherapy comprises treatment with Monalizumab.

55. The method of any one of items 24, 26, 29 and 31 , wherein said immunotherapy treatment is an anti-KIR based immunotherapy.

56. The method of item 55, wherein said anti-KIR based

immunotherapy comprises treatment with IPH4102 or Lirilumab.

57. A method of identifying a subject responsive to, or suitable to receive, treatment with a Vps34 inhibitor wherein the subject is afflicted with cancer, comprising

- providing a biological test sample originating from said subject;

- determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in said biological test sample; and

wherein, if the level of said at least one biomarker in said test sample is lower than said predetermined threshold value, said patient is identified as likely to respond to and/or suitable to receive immunostimulating treatment using a Vps34 inhibitor.

58. A method of identifying a subject responsive to, or suitable to receive, treatment with a Vps34 inhibitor wherein the subject is afflicted with cancer, comprising

- providing a biological test sample originating from said subject;

- determining the level of at least one biomarker selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in said biological test sample; and

- comparing the level of said at least one biomarker in said test sample to a predetermined threshold value, wherein, if the level of said at least one biomarker in said test sample is lower than said predetermined threshold value, said patient is identified as likely to respond to and/or suitable to receive immunostimulating treatment using a Vps34 inhibitor.

59. The method of item 57 or 58, wherein, if the level of said at least one biomarker in said test sample is lower than said predetermined threshold value, said patient is identified as suitable to receive a combination treatment using a Vps34 inhibitor and immune therapy.

60. A method according to any one of items 24, 25, 26, 27, 29, 30, 31 , 32, 57 and 58, wherein the at least one biomarker is selected from CCL5 and CXCL10.

61. The method of any one of the preceding items, wherein said Vps34 inhibitor is selected from the group consisting of:

4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-2- amine,

1 -[[5-[2-[(2-chloro-4-pyridyl)amino]pyrimidin-4-yl]-4-

(cyclopropylmethyl)pyrimidin-2-yl]amino]-2-methyl-propan-2-ol,

1 -[[4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-

2-yl]amino]-2-methyl-propan-2-ol,

(2S)-1 -[(5-chloro-3-pyridyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2- (trifluoromethyl)-3,4-dihydro-2H-pyrimido[1 ,2-a]pyrimidin-6-one,

(2S)-8-[(3R)-3-methylmorpholin-4-yl]-1 -(3-methyl-2-oxo-butyl)-2- (trifluoromethyl)-3,4-dihydro-2H-pyrimido[1 ,2-a]pyrimidin-6-one,

N-[4-[5-[(2,4-difluorophenyl)sulfonylamino]-6-methyl-3-pyridyl]-2- pyridyl]acetamide,

N-[2-chloro-4-methyl-5-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-3- pyridyl]-2,4-difluoro-benzenesulfonamide,

N-[4-[6-chloro-5-[(2,4-difluorophenyl)sulfonylamino]-4-methyl-3-pyridyl]-

2-pyridyl]cyclopropanecarboxamide,

N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2-pyridyl]acetamide,

4-(2-methyl-1 H-pyrrolo[2,3-b]pyridin-4-yl)-6-morpholino-1 H-pyridin-2- one 4-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-6-[3- (trifluoromethyl)morpholin-4-yl]-1 H-pyridin-2-one,

4-(1 H-pyrazolo[3,4-b]pyridin-4-yl)-6-[2-(trifluoromethyl)-1 -piperidyl]-1 H- pyridin-2-one,

N-[4-[2-oxo-6-[3-(trifluoromethyl)nnorpholin-4-yl]-1 H-pyridin-4-yl]-2- pyridyl]acetamide,

4-[(3R)-3-methylmorpholin-4-yl]-6-[4-pyrrolidin-1 -ylsulfonyl-2- (trifluoromethyl)piperazin-l -yl]-1 H-pyridin-2-one,

4-[(3R)-3-methylmorpholin-4-yl]-6-[4-(2-nnethylpyrazol-3-yl)sulfonyl-2- (trifluoromethyl)piperazin-l -yl]-1 H-pyridin-2-one,

6-[4-cyclopropylsulfonyl-2-(trifluoromethyl)piperazin-1-yl]-4-[(3R)-3- methylmorpholin-4-yl]-1 H-pyridin-2-one,

4-[2-[(2-methylpyrimidin-4-yl)annino]-4-pyridyl]-6-[2- (trifluoromethyl)phenyl]-1 H-pyridin-2-one,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)-1 -piperidyl]- 1 H-pyridin-2-one,

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)pyrrolidin-1 - yl]-1 H-pyridin-2-one,

4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-

2-one,

6-[2-(3-methoxyphenyl)pyrrolidin-1 -yl]-4-[(3R)-3-methylmorpholin-4-yl]- 1 H-pyridin-2-one,

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-phenyl-1 -pipendyl]-1 H- pyridin-2-one,

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2S)-2-(trifluoromethyl)-1 -piperidyl]- 1 H-pyridin-2-one,

6-(2-chlorophenyl)-1 -methyl-4-(3-methylmorpholin-4-yl)pyridin-2-one 4-[(3R)-3-methylmorpholin-4-yl]-6-[2-(trifluoromethyl)phenyl]-1 H- pyridin-2-one,

6-(2-chlorophenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2-one, and 6-(2-chloro-5-fluoro-phenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H- pyridin-2-one.

62. The method of any one of the preceding items, wherein said Vps34 inhibitor is selected from the group consisting of:

4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-2- amine,

1 -[[5-[2-[(2-chloro-4-pyridyl)amino]pyrimidin-4-yl]-4-

(cyclopropylmethyl)pyrimidin-2-yl]amino]-2-methyl-propan-2-ol,

1 -[[4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-

2-yl]amino]-2-methyl-propan-2-ol,

N-[4-[6-chloro-5-[(2,4-difluorophenyl)sulfonylamino]-4-methyl-3-pyridyl]-

2-pyridyl]cyclopropanecarboxamide,

N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2-pyridyl]acetamide, 4-(2-methyl-1 H-pyrrolo[2,3-b]pyridin-4-yl)-6-morpholino-1 H-pyridin-2- one,

4-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-6-[3- (trifluoromethyl)morpholin-4-yl]-1 H-pyridin-2-one,

N-[4-[2-oxo-6-[3-(trifluoromethyl)morpholin-4-yl]-1 H-pyridin-4-yl]-2- pyridyl]acetamide,

4-[(3R)-3-methylmorpholin-4-yl]-6-[4-(2-methylpyrazol-3-yl)sulfonyl-2- (trifluoromethyl)piperazin-l -yl]-1 H-pyridin-2-one,

6-[4-cyclopropylsulfonyl-2-(trifluoromethyl)piperazin-1-yl]-4-[(3R)-3- methylmorpholin-4-yl]-1 H-pyridin-2-one, 4-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-6-[2- (trifluoromethyl)phenyl]-1 H-pyridin-2-one,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-

2-one,

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-phenyl-1 -piperidyl]-1 H- pyridin-2-one,

6-(2-chlorophenyl)-1 -methyl-4-(3-methylmorpholin-4-yl)pyridin-2-one

4-[(3R)-3-methylmorpholin-4-yl]-6-[2-(trifluoromethyl)phenyl]-1 H- pyridin-2-one,

6-(2-chlorophenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2-one, and

6-(2-chloro-5-fluoro-phenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H- pyridin-2-one.

63. The method of any preceding item, wherein said Vps34 inhibitor is selected from the group consisting of:

N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2-pyridyl]acetamide,

4-(2-methyl-1 H-pyrrolo[2,3-b]pyridin-4-yl)-6-morpholino-1 H-pyridin-2- one,

4-(1 H-pyrazolo[3,4-b]pyridin-4-yl)-6-[2-(trifluoromethyl)-1 -piperidyl]-1 H- pyridin-2-one, N-[4-[2-oxo-6-[3-(trifluoromethyl)morpholin-4-yl]-1 H-pyridin-4-yl]-2- pyridyl]acetamide,

4-[2-[(2-methylpyrimidin-4-yl)amino]-4-pyridyl]-6-[2- (trifluoromethyl)phenyl]-1 H-pyridin-2-one,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-

2-one,

6-(2-chlorophenyl)-1 -methyl-4-(3-methylmorpholin-4-yl)pyridin-2-one,

6-(2-chlorophenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2-one, and

64. The method according to any one of items 1 -61 , wherein said Vps34 inhibitor is selected from the group consisting of:

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-phenyl-1 -piperidyl]-1 H- pyridin-2-one, 4-[(3R)-3-methylmorpholin-4-yl]-6-[2-(trifluoromethyl)phenyl]-1 H- pyridin-2-one,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

(2S)-1 -[(5-chloro-3-pyridyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2- (trifluoromethyl)-3,4-dihydro-2H-pyrimido[1 ,2-a]pyrimidin-6-one, and

4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)-1 -piperidyl]- 1 H-pyridin-2-one.

65. The method of any preceding item, wherein said Vps34 inhibitor is 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)-1 -piperidyl]-1 H- pyridin-2-one.

66. Use of at least one cytokine selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy as a biomarker of inhibition of Vps34 in a cancer cell.

67. Use of at least one cytokine selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy as a biomarker of inhibition of Vps34 in a cancer cell.

68. Use according to item 66 or 67, wherein the biomarker is CCL5 and/or CXCL10.

69. Use according to any one of items 66, 67 and 68, wherein at least two biomarkers selected from said group are detected.

70. A biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy for use in a method of treating cancer.

71. A biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy for use in a method of treating cancer. 72. The biomarker for use according to item 70 or 71 , wherein the biomarker is CCL5 and/or CXCL10.

73. A combination of biomarkers selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy for use in a method of treating cancer.

74 A combination of biomarkers selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy for use in a method of treating cancer.

75. A combination of CCL5 and CXCL10 for use as biomarkers in a method of treating cancer.

76. The method of any one of items 24, 26, 29 and 31 , wherein said immunotherapy comprises treatment with a STING agonist.

77. The method of item 76, wherein said immunotherapy treatment comprises treatment with MK-1454. LIST OF REFERENCES

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Claims

CCL5, CXCL9, CXCL10, CXCL11 and IFNy, preferably selected from CCL5 and CXCL10, wherein an elevated level of said at least one biomarker in said test sample, relative to a reference level, indicates immunostimulation.

3. The method of claim 1 or 2, wherein the mRNA expression level of said at least one biomarker is determined.

4. The method of claim 1 or 2, wherein the protein concentration of said at least one biomarker in said sample is determined.

5. The method of any one of the preceding claims, comprising i) providing at least one test sample; and

ii) in vitro analysing said test sample to determine the level of said at least one biomarker, wherein an elevated level of said at least one biomarker, relative to a reference level, indicates immunostimulation.

6. The method of any one of the preceding claims, wherein the reference level is based on a reference sample from a source that has not been exposed to or subjected to treatment with a Vps34 inhibitor,

7. The method of claim 6, comprising in vitro analysing said reference sample and said test sample to determine the level of at least one biomarker in each of said samples, and wherein an elevated level of said at least one biomarker in the test sample relative to the level of the biomarker in the reference sample indicates immunomostimulation in said source.

8. The method of claim 5, comprising

ii) in vitro analysing said biological reference sample and said biological test sample to determine the level of at least one biomarker in each of said samples, and wherein an elevated level of said at least one biomarker in the biological test sample relative to the biological reference sample indicates immunostimulation in said source treated with a Vps34 inhibitor.

9. The method of any one of the preceding claims, wherein the test sample comprises a material selected from the group consisting of: cell culture supernatant, tumour plasma, and bodily fluids such as blood, blood plasma and urine, and preferably is blood plasma.

10. The method of any one of the preceding claims, wherein the reference level is based on a reference sample comprising a material selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine, and preferably is blood plasma.

11. The method of claim 9 or 10, wherein the test sample is blood plasma or tumour plasma, preferably blood plasma; and the reference sample is blood plasma or tumour plasma, preferably blood plasma.

12. The method according to any one of the preceding claims, wherein said source is a cancer cell or tumour maintained in vitro.

14. A method of determining and/or predicting the immunostimulating effect of treatment with Vps34 inhibitor in a subject afflicted with cancer, wherein said treatment comprises administration of a Vps34 inhibitor to said subject, said method comprising

15. The method of claim 13 or 14, wherein said reference level is based on a reference sample obtained from said subject prior to said treatment.

16. The method of any one of claims 13 to 15, wherein the test sample and/or the reference sample is selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine.

17. The method of claim 15, wherein the test sample is blood plasma or tumour plasma, preferably blood plasma; and the biological reference sample is blood plasma or tumour plasma, preferably blood plasma.

18. The method of claim 15, comprising the steps of

b) optionally administering a Vps34 inhibitor to said subject;

c) providing a test sample originating from said subject after said treatment;

19. The method of claim 13 or 14, wherein in step i) the mRNA expression level of said at least one biomarker is determined in said test sample.

20. The method of claim 13 or 14, wherein in step i) the protein concentration of said at least one biomarker is determined in said test sample.

21. The method of claim 15, wherein the mRNA expression level of said at least one biomarker is determined in said reference sample.

22. The method of claim 15, wherein the protein concentration of said at least one biomarker is determined in said reference sample.

23. The method of claim 13 or 14, wherein the at least one biomarker is selected from CCL5 and CXCL10.

24. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor;

ii) comparing the level of said biomarker in said test sample to a predetermined reference level; and

25. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor;

ii) comparing the level of said biomarker in said test sample to a predetermined reference level; and iii) if the level of said biomarker in said test sample is elevated relative to the reference level, selecting continued treatment with the Vps34 inhibitor.

26. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor; ii) comparing the level of said biomarker in said test sample to a predetermined reference level; and

iii) if the level of said biomarker in said test sample is elevated relative to the reference level, selecting immunotherapy as a further treatment optionally in combination with treatment with the Vps34 inhibitor.

27. A method of selecting a treatment regimen for a subject afflicted with cancer who has received treatment with a Vps34 inhibitor, comprising i) determining the level of at least one biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy in a test sample originating from said subject having received treatment with a Vps34 inhibitor; ii) comparing the level of said biomarker in said test sample to a predetermined reference level; and

28. The method of any one of claims 24 to 27, wherein step i) further comprises determining the level of said at least one biomarker in a reference sample obtained from said subject prior to treatment with the Vps34 inhibitor, and the predetermined reference level corresponds to the level of the biomarker in the reference sample.

29. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject; (b) obtaining a test sample from said subject;

30. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

31. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

32. A method of treating a subject afflicted with cancer, comprising

(a) administering a Vps34 inhibitor to said subject;

(b) obtaining a test sample from said subject;

33. The method of any one of claims 29 to 32, comprising, prior to the step of (a) administering a Vps34 inhibitor to said subject, obtaining a reference sample from said subject, and further comprising determining the level of said at least one biomarker in said reference sample, and wherein said predetermined reference level represents the level of said at least one biomarker determined in said reference sample.

34. The method of any one of claims 29 to 32, wherein said subject is subjected to further treatment with the Vps34 inhibitor simultaneously, intermittently or sequentially with said immunotherapy.

35. The method of any one of the claims 24 to 27 and 29 to 32, wherein the mRNA expression level and of said at least one biomarker is determined in said test sample.

36. The method of any one of claims 24 to 27 and 29 to 32, wherein the protein concentration of said at least one biomarker is determined in said test sample.

37. The method of claim 28 or 33 wherein the mRNA expression level of said at least one biomarker is determined in said reference sample.

38. The method of claim 28 or 33 wherein the protein concentration of said at least one biomarker is determined in said reference sample.

39. The method of any one of claims 24 to 27 and 29 to 32, wherein the biological test sample is selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine.

40. The method of claims 28 or 33, wherein the biological reference sample is selected from the group consisting of: cell culture supernatant, tumour plasma, or bodily fluid such as blood, blood plasma and urine.

41. The method of claim 40, wherein the biological test sample is blood plasma or tumour plasma, preferably blood plasma; and the biological reference sample is blood plasma or tumour plasma, preferably blood plasma.

42. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment is an immune checkpoint blockade-based therapy.

43. The method of claim 42, wherein said immunotherapy is anti-PD-1 or PD-L1 based immunotherapy or macrophage immune checkpoint based immunotherapy.

44. The method of claim 43, wherein said anti-PD-1 or PD-L1 based immunotherapy comprises treatment with at least one agent selected from the group consisting of Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Tislelizumab, Sintilimab or said macrophage immune checkpoint based immunotherapy comprises treatment with at least one agent selected from the group consisting of an anti-CD47 antibody and an anti-SIRPalpha antibody.

45. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment is an anti-CTLA-4 based immunotherapy.

46. The method of claim 45, wherein said anti-CTLA-4 based immunotherapy comprises treatment with an agent selected from Ipilimumab and Tremelimumab.

47. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment comprises treatment with an oncolytic virus.

48. The method of claim 47, wherein said oncolytic virus is selected from Oncorine, Talimogene laherparepvec and Pelareorep.

49. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment is a CAR-T cell therapy.

50. The method of claim 49, wherein said CAR-T cell therapy comprises treatment with Tisagenlecleucel or Axicabtagene ciloleucel.

51. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment comprises treatment with a CD3-targeted bispecific antibody.

52. The method of claim 51 , wherein said CD3-targeted bispecific antibody is Blinatumomab or duvortuxizumab.

53. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment is an anti-NKG2A based immunotherapy.

54. The method of claim 53, wherein said anti-NKG2A based

immunotherapy comprises treatment with Monalizumab.

55. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy treatment is an anti-KIR based immunotherapy.

56. The method of claim 55, wherein said anti-KIR based

immunotherapy comprises treatment with IPH4102 or Lirilumab.

57. A method according to any one of claims 24 to 27 and 29 to 32, wherein the at least one biomarker is selected from CCL5 and CXCL10.

58. The method of any one of the preceding claims, wherein said Vps34 inhibitor is selected from the group consisting of:

4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-2- amine,

1 -[[5-[2-[(2-chloro-4-pyridyl)amino]pyrimidin-4-yl]-4-

(cyclopropylmethyl)pyrimidin-2-yl]amino]-2-methyl-propan-2-ol,

1 -[[4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-

2-yl]amino]-2-methyl-propan-2-ol,

N-[4-[6-chloro-5-[(2,4-difluorophenyl)sulfonylamino]-4-methyl-3-pyridyl]-

2-pyridyl]cyclopropanecarboxamide, N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2-pyridyl]acetamide, 4-(2-methyl-1 H-pyrrolo[2,3-b]pyridin-4-yl)-6-morpholino-1 H-pyridin-2- one,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-

2-one,

6-(2-chlorophenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2-one, and

59. The method of any one of the preceding claims, wherein said Vps34 inhibitor is selected from the group consisting of:

4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-2- amine,

1 -[[5-[2-[(2-chloro-4-pyridyl)amino]pyrimidin-4-yl]-4-

(cyclopropylmethyl)pyrimidin-2-yl]amino]-2-methyl-propan-2-ol,

1 -[[4-(cyclopropylmethyl)-5-[2-(4-pyridylamino)pyrimidin-4-yl]pyrimidin-

2-yl]amino]-2-methyl-propan-2-ol,

N-[4-[6-chloro-5-[(2,4-difluorophenyl)sulfonylamino]-4-methyl-3-pyridyl]-

2-pyridyl]cyclopropanecarboxamide,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-

2-one,

6-(2-chlorophenyl)-1 -methyl-4-(3-methylmorpholin-4-yl)pyridin-2-one

6-(2-chlorophenyl)-4-[(3R)-3-methylmorpholin-4-yl]-1 H-pyridin-2-one, and

60. The method of any preceding claim, wherein said Vps34 inhibitor is selected from the group consisting of:

4-[2-[(2-methylpyrimidin-4-yl)annino]-4-pyridyl]-6-[3- (trifluoromethyl)nnorpholin-4-yl]-1 H-pyridin-2-one,

4-(1 H-pyrazolo[3,4-b]pyridin-4-yl)-6-[2-(trifluoronnethyl)-1 -piperidyl]-1 H- pyridin-2-one,

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

4-[(3R)-3-methylmorpholin-4-yl]-6-(2-phenylpyrrolidin-1 -yl)-1 H-pyridin-

2-one,

6-(2-chlorophenyl)-1 -methyl-4-(3-methylmorpholin-4-yl)pyridin-2-one,

61. The method according to any one of claims 1 -58, wherein said Vps34 inhibitor is selected from the group consisting of:

methyl N-[4-[2-(2-chlorophenyl)-6-oxo-1 H-pyridin-4-yl]-2- pyridyl]carbamate,

62. The method of any one of the preceding claims, wherein said Vps34 inhibitor is 4-[(3R)-3-methylmorpholin-4-yl]-6-[(2R)-2-(trifluoromethyl)- 1 -piperidyl]-1 H-pyridin-2-one.

63. Use of at least one cytokine selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy as a biomarker of inhibition of Vps34 in a cancer cell.

64. Use of at least one cytokine selected from the group consisting of CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy as a biomarker of inhibition of Vps34 in a cancer cell.

65. Use according to claim 63 or 60, wherein the biomarker is CCL5 and/or CXCL10.

66. Use according to any one of claims 63, 64 and 65, wherein at least two biomarkers selected from said group are detected.

67. A biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy for use in a method of treating cancer.

68. A biomarker selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy for use in a method of treating cancer.

69. The biomarker for use according to claim 67 or 68, wherein the biomarker is CCL5 and/or CXCL10.

70. A combination of biomarkers selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10 and IFNy for use in a method of treating cancer.

71. A combination of biomarkers selected from CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11 and IFNy for use in a method of treating cancer.

72. A combination of CCL5 and CXCL10 for use as biomarkers in a method of treating cancer.

73. The method of any one of claims 24, 26, 29 and 31 , wherein said immunotherapy comprises treatment with a STING agonist.

74. The method of claim 73, wherein said immunotherapy treatment comprises treatment with MK-1454.