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Figure S1: Hierarchically clustered heat map of mutations in the cell line panel. Figure S2: Heat map of median-centered CCLE cell line basal mRNA. Figure S3: Changes in pAKTSer473 quantified by ELISA.

Table S1: KRAS mutations in colorectal, lung and pancreatic cell lines. Table S2: Assay variability. Table S3: Significant changes in phosphoproteins seen in more than 50% of cell lines. Table S4: The GI50 concentrations of the two pan-PI3K inhibitors pictilisib and buparlisib used in validation experiments in Figure 4B.

Cell lines, culture medium, drug concentrations, cell culture, methods for producing Figure 1

Supplementary Note

Figure S2. Continuation of IMP following radiological detection of asymptomatic DILD resulting in clinical deterioration.

Table S1. Frequency of DILD as per category

Purpose:Drug-induced interstitial lung disease (DILD) is a rare, but potentially fatal toxicity. Clinical and radiological features of DILD in the early experimental setting are poorly described.Patients and Methods:A total of 2,499 consecutive patients with advanced cancer on phase I clinical trials were included. DILD was identified by a dedicated radiologist and investigators, categorized per internationally recognized radiological patterns, and graded per Common Terminology Criteria for Adverse Events (CTCAE) and the Royal Marsden Hospital (RMH) DILD score. Clinical and radiological features of DILD were analyzed.Results:Sixty patients overall (2.4%) developed DILD. Median time to onset of DILD was 63 days (range, 14–336 days). A total of 45% of patients who developed DILD were clinically asymptomatic. Incidence was highest in patients receiving drug conjugates (7.4%), followed by inhibitors of the PI3K/AKT/mTOR pathway (3.9%). The most common pattern seen was hypersensitivity pneumonitis (33.3%), followed by nonspecific interstitial pneumonia (30%), and cryptogenic organizing pneumonia (26.7%). A higher DILD score [OR, 1.47, 95% confidence interval (CI), 1.19–1.81; P < 0.001] and the pattern of DILD (OR, 5.83 for acute interstitial pneumonia; 95% CI, 0.38–90.26; P = 0.002) were significantly associated with a higher CTCAE grading. The only predictive factor for an improvement in DILD was an interruption of treatment (OR, 0.05; 95% CI, 0.01–0.35; P = 0.01).Conclusions:DILD in early-phase clinical trials is a toxicity of variable onset, with diverse clinical and radiological findings. Radiological findings precede clinical symptoms. The extent of the affected lung parenchyma, scored by the RMH DILD score, correlates with clinical presentation. Most events are low grade, and improve with treatment interruption, which should be considered early.

Figure S1A. Correlation of patterns of DILD as per ATS/ERS classification and clinical severity. AIP correlated significantly with worse severity (P=0.002). NSIP, non-specific interstitial pneumonia; COP, cryptogenic organizing pneumonia; AIP, acute interstitial pneumonia; HP, hypersensitivity pneumonitis. Figure S1B. A higher RMH DILD score predicts for increasing clinical severity of DILD as per CTCAE grading. Shown are CTCAE grading and RMH DILD Score at the time of DILD detection. Box plot mapping RMH DILD Scores against CTCAE grading showing statistically significant worsening of CTCAE grading and symptoms with higher RMH DILD score (P=0.004); DILD was confirmed to be the cause of death in one patient who had an initial RMH DILD score of 5 and was asymptomatic with an excellent partial response to therapy and so was continued on therapy. This patient then developed symptomatic DILD with a worsening RMH DILD score of 8, deteriorated and died. RMH DILD score, Royal Marsden Hospital Score for drug induced interstitial lung disease; CTCAE, Common Terminology Criteria for Adverse Events Version 5.0. Figure S2. Continuation of IMP following radiological detection of asymptomatic DILD resulting in clinical deterioration. Images from a patient with breast cancer treated with an investigational drug-conjugate. Patient had no prior lung disease and no lung metastases at baseline. Cartoons (left) show RMH DILD scoring of matched sagittal radiographic images (right). The first row shows initial development of radiographic changes after 2 cycles of treatment when patient was asymptomatic (RMH DILD score of 5) and otherwise had a remarkable RECIST PR. With a further cycle of treatment, the changes had significantly worsened (RMH DILD score of 8), with development of rapidly progressive clinical symptoms which was ultimately fatal despite treatment with antibiotics, steroids, and respiratory support.

Background PTEN loss of function is frequent in GBM correlating with poor prognosis, impaired antitumor responses and reduced efficacy of Immune Checkpoint Inhibitors (ICI). Ipat is a potent, selective, small-molecule inhibitor of Akt. Ipat efficiently depletes FOXP3+ regulatory T cells from the tumor microenvironment (TME) resulting in increased infiltration of effector T cells in solid tumors (Lopez 2020, AACR). We hypothesize that Akt inhibition in PTEN loss glioblastomas may deplete the TME of suppressive immune cells, and render malignant brain tumors more responsive to ICIs. Methods Relapsed WHO grade IV GBM pts with stable neurological symptoms ≥5 days prior to enrollment, requiring Results 23 recurrent GBM pts (median age 55 yrs (25-71 yrs; 74% male) were enrolled. Median ECOG PS 1. All pts had surgery followed by radical-chemoradiotherapy. Median prior lines of therapy 1 (range 1-4). 15 pts had PTEN loss by IHC (H 6 cycles) in 19 efficacy evaluable pts was 32% (6/19 overall) and 28.6% (4/14) for pts with PTEN loss. Multiplex IHC of archival samples (n=19) showed PTEN loss tumors had significantly greater numbers of CD3+ infiltrating T-lymphocytes within the TME compared to PTEN wild type tumors (median 53.64/mm2 vs 1.94/mm2; p=0.0021). Within the PTEN loss cohort (n=13), pts with clinical benefit had significantly higher baseline number of CD8+ effector T cells as compared to non-responders (median 46.50/mm2 vs 8.21/mm2; p=0.0336). Two pts with PTEN loss proceeded to re-resection on trial. One exceptional responder, a 58yr male refractory to radical chemo-radiotherapy and Bevacizumab had a resection of enhancing disease after 5 cycles that showed >70% depletion of CD4+ T regulatory cells with an increase in CD8+ lymphocyte infiltration and no residual evidence of tumor (pathological CR). In contrast, a non-responding patient who proceeded to debulking surgery had no change in infiltration of CD8+ lymphocytes, but a marked increase (>27-fold) in CD4+ regulatory T cells. Conclusion The RP2D of 400mg Ipa OD + 1200mg A Q3W was well tolerated in GBM pts. Early efficacy signals were detected with PTEN loss being a promising predictive biomarker for response to combination. Citation Format: Crescens Tiu, Wing Hing Yau, Liam C. Welsh, Timothy L. Jones, Anna Zachariou, Toby Prout, Mona Parmar, Alison J. Turner, Robert W. Daly, Christina Yap, Mateus Crespo, Bora Gurel, Ruth M. Riisnaes, Shaun A. DeCordova, Karen E. Swales, Udai Banerji, Anna R. Minchom, Adam Sharp, Johann S. de Bono, Juanita S. Lopez. Preliminary evidence of antitumor activity of Ipatasertib (Ipat) and Atezolizumab (A) in glioblastoma (GBM) patients (pts) with PTEN loss in the Phase 1 Ice-CAP trial (NCT03673787) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT093.

It is increasingly appreciated that drug response to different cancers driven by the same oncogene is different and may relate to differences in re-wiring of signal transduction. We aimed to study differences in dynamic signaling changes within mutant KRAS (KRAS(MT)), non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC) cells. We used an antibody-based phosphoproteomic platform to study changes in 50 phosphoproteins caused by seven targeted anticancer drugs in a panel of 30 KRAS(MT) cell lines and cancer cells isolated from 10 patients with KRAS(MT) cancers. We report for the first time significant differences in dynamic signaling between CRC and NSCLC cell lines exposed to clinically relevant equimolar concentrations of the pan-PI3K inhibitor pictilisib including a lack of reduction of p-AKTser473 in CRC cell lines (P = 0.037) and lack of compensatory increase in p-MEK in NSCLC cell lines (P = 0.036). Differences in re-wiring of signal transduction between tumor types driven by KRAS(MT) cancers exist and influence response to combination therapy using targeted agents.